PCR PRODUCTS. Table of Contents

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2 Introduction to takara PCR Enzymes How to Select the Best PCR Enzyme... 2 Introduction to Takara s Patented LA PCR Technology Guide to Takara Polymerases... 4 high fidelity PCR PrimeSTAR HS DNA Polymerase... 6 PrimeSTAR HS DNA Polymerase with GC Buffer... 6 PrimeSTAR HS (Premix)... 6 PrimeSTAR GXL DNA Polymerase NEW... 7 PrimeSTAR Max DNA Polymerase NEW... 7 PCR products RACE Core Set, 3'-Full and 5'-Full BcaBEST RNA PCR Kit Real time PCr (qpcr) kits EASY Dilution Solution (for real time PCR) SYBR Green I Detection (qpcr Products) SYBR Premix Ex Taq (Tli RNase H Plus) NEW SYBR Premix Ex Taq II (Tli RNaseH Plus) NEW SYBR Premix DimerEraser (Perfect Real Time) Transgene Detection Primer Set for Real Time (Mouse) Probe Detection (qpcr Products) Premix Ex Taq (Probe qpcr) NEW PCR PRODUCTS High Performance PCR TaKaRa Ex Taq DNA Polymerase TaKaRa Ex Taq... 8 EmeraldAmp Max PCR Master Mix NEW... 9 Premix Taq (Ex Taq Version)... 9 TaKaRa Ex Taq, Hot-Start Version PerfectShot Ex Taq (Loading Dye Mix) EmeraldAmp Max HS PCR Master Mix NEW Taq Antibody TaKaRa LA Taq DNA Polymerase TaKaRa LA Taq One-Shot LA PCR Mix LA PCR Kit Version TaKaRa LA Taq Hot-Start Version LA PCR Genome DNA Set TaKaRa LA Taq with GC Buffers HIGH SPEED PCR SapphireAmp Fast PCR Master Mix NEW SpeedSTAR HS DNA Polymerase TaKaRa Z-Taq products for routine PCR TaKaRa Taq and Premix Taq (TaKaRa Taq Version) EmeraldAmp GT PCR Master Mix NEW PCR Amplification Kit TaKaRa Taq Hot-Start Version EpiScope MSP Kit DNA Fragmentation Kit Deoxynucleotide Triphosphates (dntps) RT-PCR PrimeScript RT PCR Kit PrimeScript 1 st Strand cdna Synthesis Kit PrimeScript II 1 st Strand cdna Synthesis Kit RNA LA PCR Kit, Version RNA PCR Kit, Version One Step RNA PCR Kit mrna Selective PCR Kit Version REAL TIME RT-PCR One Step SYBR PrimeScript qrt-pcr Kit One Step SYBR PrimeScript qrt-pcr Kit II One Step PrimeScript qrt-pcr Kit PrimeScript RT Reagent Kit CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) Cell Amp Whole Transcriptome Amplification Kit (Real Time) CellAmp Direct RNA Prep Kit for Real Time PCR and Protein Analysis PrimerArray Series FOOD PATHOGEN Detection & screening Kits PCR Mycoplasma Detection Set O-157 (Verocytotoxin Genes) One-Shot PCR Screening Kit.40 O-157 (Verocytotoxin Genes) PCR Screening Set O-157 (Verocytotoxin Genes) One-Shot PCR Typing Kit O-157 (Verocytotoxin Genes) PCR Typing Kit Multiplex PCR O-157/Verocytotoxin Genes Detection Kit. 42 Bacillus anthracis PCR Detection Kit Bacteria Screening PCR Kit b-globin (human) Primer Set (Control Primer for PCR) Campylobacter (cdt gene) PCR Detection and Typing Kit PCR Probes and Primer sets PCR Human Papillomavirus Detection Set PCR Human Papillomavirus Typing Set Enzyme Set A for the HPV Typing Set Bacterial Pathogens PCR Positive Control Templates Bacterial Pathogens PCR Primer Sets Table of Contents 1

3 1PCR PRODUCTS How to Select the Best PCR Enzyme How To Select the Best PCR Enzyme 2

4 Introduction to Takara s Patented LA PCR Technology Taq Polymerase, the workhorse enzyme most commonly used for PCR, has several limitations. It does not possess a proofreading (3 g5 exonuclease) activity, and so has a relatively high rate of base misincorporations (error rate). At the end of a typical 30-cycle PCR reaction, a significant portion of the products generated with Taq Polymerase will contain one or more errors, especially in longer products. Because Taq Polymerase tends to fall off DNA templates at the sites of these misincorporations, both product yield and length are restricted. Long and Accurate PCR Technology (LA PCR) provides a solution to the problems intrinsic to the use of conventional Taq Polymerase. LA PCR Technology involves mixing Taq Polymerase with a small amount of a proofreading polymerase, producing an enzyme mix with performance characteristics (fidelity, yield, length, reproducibility) superior to either enzyme alone. Takara Bio owns the worldwide patent on Long and Accurate (LA) PCR, and provides the largest available selection of high-performance PCR reagents and kits based on this technology. Ex Taq Polymerase offers high sensitivity, increased product yield and length, and improved reproducibility and fidelity over Taq Polymerase. LA Taq Polymerase can synthesize products up to 48 kb in length with fidelity 6.5X better than Taq Polymerase, and requires less optimization than other long PCR polymerases. LA Taq Polymerase with GC Buffer is optimized for amplification of GC-rich templates, and SpeedSTAR HS DNA Polymerase is highly processive, with reaction times up to 5X faster than Taq. Premix and Hot-Start versions of Ex Taq and LA Taq are available, as well as qpcr, RT-PCR, RACE, cloning, mutagenesis, and screening kits. PCR PRODUCTS 1 Principle of LA PCR Technology. The key to LA PCR technology is the enzyme mix. Both Takara Ex Taq and Takara LA Taq are thermostable DNA polymerases which possess 3 g5 exonuclease, or proofreading activity. This 3 g5 exonuclease activity removes misincorporated bases, allowing subsequent product extension to proceed smoothly and efficiently, making amplification of long DNA fragments possible. How To Select the Best PCR Enzyme 3

5 Guide to TaKaRa 1PCR PRODUCTS Guide to Takara Polymerases Application Routine PCR High Efficiency PCR High Fidelity PCR Long PCR Polymerase* Amplification Efficiency Product Size w/ λdna (Average/Max) TaKaRa Taq * (R001A) ++ 6 kb/12 kb Premix Taq (R004A) ++ 6 kb/12 kb TaKaRa Taq HS* (R007A) ++ 6 kb/12 kb Premix Taq HS (R028A) ++ 6 kb/12 kb EmeraldAmp GT PCR Master Mix* (RR310A) ++ 6 kb/12 kb TaKaRa Ex Taq * (RR001A) kb/30 kb Premix Ex Taq (RR003A) kb/30 kb TaKaRa Ex Taq HS* (RR006A) kb/30 kb Premix Ex Taq HS (RR030A) kb/30 kb PerfectShot Ex Taq (RR005A) kb/30 kb EmeraldAmp Max PCR Master Mix* (RR320A) kb/30 kb EmeraldAmp Max HS PCR Master Mix* (RR330A) kb/30 kb PrimeSTAR MAX* (R045A) ++++ up to 6 kb PrimeSTAR GXL* (R050A) kb PrimeSTAR HS* (R010A) +++ up to 20 kb PrimeSTAR HS with GC Buffers (R044A) +++ up to 10 kb PrimeSTAR HS, Premix (R040A) +++ up to 10 kb TaKaRa LA Taq * (RR002A) kb/48 kb TaKaRa LA Taq w/gc Buffers (RR02AG) kb/48 kb LA PCR Kit, V.2.1 (RR013A) kb/48 kb One-Shot LA PCR Mix V.2.0 (RR004) kb/48 kb TaKaRa LA Taq HS* (RR042A) kb/48 kb Fast PCR Real Time PCR SpeedSTAR HS* (RR070A) kb/30 kb SapphireAmp Fast PCR Master Mix* (RR350A) kb/30 kb SYBR Premix DimerEraser * (RR091A) +++ _ SYBR Premix Ex Taq *(Tli RNaseH Plus) (RR420A) ++++ _ SYBR Premix Ex Taq * II (Tli RNaseH Plus) (RR820A) ++++ _ * Sample Available Premix Ex Taq *(Probe qpcr) (RR390A) ++++ _ g Unit Definition One unit is the amount of enzyme that will incorporate 10 nmol of dntp into acid-insoluble products in 30 min. at 74 C with activated salmon sperm DNA as the template-primer. Purity Nicking activity, endonuclease, and exonuclease activity were not detected after the incubation of 0.6 µg of double-stranded supercoiled pbr322 DNA, 0.6 µg of λ DNA, or 0.6 µg of λ-hind III digest with 10 units of enzyme for 1 hour at 74 C. 4

6 PCR Polymerases Product Size w/ Human Genomic DNA (Average/ Max) Hot Start PCR Fidelity Proofreading Activity Robustness GC-Rich Templates Real Time PCR (qpcr) Processing Speed Terminal Transferase Activity (3 -A overhang) 2 kb/4 kb No +** No ++ + _ 1 kb/min Yes 2 kb/4 kb No +** No _ 1 kb/min Yes 2 kb/4 kb Yes +** No kb/min Yes 2 kb/4 kb Yes +** No kb/min Yes 2 kb/4 kb No +** No _ 1 kb/min Yes 10 kb/20 kb No ++** Yes ++ + _ 1-2 kb/min Yes 10 kb/20 kb No ++** Yes _ 1-2 kb/min Yes 10 kb/20 kb Yes ++** Yes kb/min Yes 10 kb/20 kb Yes ++** Yes kb/min Yes 10 kb/20 kb No ++** Yes ++ + _ 1-2 kb/min Yes 10 kb/20 kb No ++** Yes _ 1-2 kb/min Yes 10 kb/20 kb Yes ++** Yes _ 1-2 kb/min Yes up to 6 kb Yes ++++ Yes _ 1 kb/5-10 sec No (blunt end) 30 kb Yes +++ Yes _ 1 kb/10 sec No (blunt end) up to 8.5 kb Yes ++++# Yes _ 1-2 kb/min No (blunt end) up to 5 kb Yes ++++# Yes _ 1-2 kb/min No (blunt end) up to 5 kb Yes ++++# Yes _ 1-2 kb/min No (blunt end) 20 kb/30 kb No +++ ** Yes ++ + _ 1-2 kb/min Yes + (20 kb/30 kb) No +++ ** Yes _ 1-2 kb/min Yes + 20 kb/30 kb No +++** Yes _ 1-2 kb/min Yes + 20 kb/30 kb No +++** Yes _ 1-2 kb/min Yes + 20 kb/30 kb Yes +++** Yes ++ + _ 1-2 kb/min Yes + 10 kb/ 20 kb Yes ++** Yes _ 6 kb/min Yes 10 kb/ 20 kb Yes ++** Yes _ 6kb/min Yes _ Yes ++** Yes _ Yes _ Yes ++** Yes _ Yes _ Yes ++** Yes _ Yes _ Yes ++** Yes _ Yes PCR PRODUCTS 1 Guide to Takara Polymerases * All of Takara s PCR polymerases are provided with dntps and buffer. They guarantee low DNA enzyme contamination ( 10 fg). + T-vector cloning efficiency diminishes as the length of the PCR product to be cloned increases above 5 kb. Dye added "Load N Go" Premixes. When used with GC Buffer I. When amplifying GC-rich templates, the fidelity is reduced. ** All fidelity determined by using the Kunkel method. # Fidelity determined by direct sequencing Best +++ Good ++ Average + Poor 5

7 1PCR PRODUCTS High Fidelity PCR Enzymes PrimeSTAR HS DNA Polymerase PrimeSTAR HS DNA Polymerase Cat.# R010A 250 U PrimeSTAR HS DNA Polymerase Cat.# R010B 1,000 U (4 250 U) High Fidelity PCR Amplification of cdna Libraries cdna Cloning for Protein Expression Site-Directed Mutagenesis and Mutant Genotyping (e.g., SNP analysis) Features High Accuracy: A strong exonuclease activity results in an extremely low error rate of only 15 errors per 480,000 bp High Efficiency: Amplification efficiency higher than Taq Polymerase; excellent performance even on GC-rich templates Robust Amplification: Tolerance to varying reaction conditions means a single PCR cycling protocol can be used to amplify products of varying sizes Extended Product Length: Amplify targets of up to 8.5 kb on human genomic DNA, 10 kb on E. coli genomic DNA, and 20 kb on l DNA Fast Reaction Times: Increased priming efficiency results in rapid annealing times High Specificity: Antibody-mediated hot-start prevents false initiation events during reaction assembly due to mispriming or primer digestion PrimeSTAR HS DNA Polymerase is a novel high fidelity PCR enzyme which provides maximum fidelity as well as extended product length (8.5 kb for human genomic DNA; 20 kb for l DNA). Targeted for demanding cloning (e.g., amplification of PrimeSTAR HS DNA Polymerase with GC Buffer PrimeSTAR HS DNA Polymerase with GC Buffer Cat.# R044A 250 U PrimeSTAR HS DNA Polymerase with GC Buffer Cat.# R044B 1,000 U (4 250 U) High Fidelity PCR with GC-Rich Templates Amplification of cdna Libraries cdna Cloning for Protein Expression PrimeSTAR HS DNA Polymerase with GC Buffer was developed for high-fidelity amplification of high-gc ($ 75%) templates. PrimeSTAR HS is a unique high fidelity DNA polymerase, offering both maximal accuracy and higher amplification efficiency than Taq Polymerase. The GC buffer formulation facilitates robust extension through even very high GC regions efficiently and accurately. Inclusion of a monoclonal antibody suppresses both the DNA polymerase and 3 g5 exonuclease activities prior to the first denaturing step, preventing false initiation events during cdna libraries) and sequencing applications, it is extremely accurate. The fidelity of PrimeSTAR HS DNA Polymerase is calculated by direct sequence analysis, which provides a more realistic estimate of the actual frequency of errors than indirect phenotypic analysis (i.e., laci) used by other suppliers. These indirect analyses are prone to error due to lethal and silent mutations. PrimeSTAR HS DNA Polymerase also offers excellent amplification efficiency and shortened reaction times. The antibody-mediated hot-start formulation prevents false initiation events during reaction assembly due to mispriming or primer digestion, lowering background. PrimeSTAR HS DNA Polymerase possesses extremely high priming efficiency. Therefore highly specific amplification can be achieved using a short annealing time, (e.g., 5 or 15 seconds). Kit Components R010A PrimeSTAR HS DNA Polymerase 100 µl 5X PrimeSTAR Buffer (Mg 2+ ) 2 1 ml dntp Mixture 800 µl 20 C Purity Nicking activity, endonuclease and exonuclease activity are not detected after the incubation of 0.6 µg of supercoiled pbr322 DNA or 0.6 µg of λ-hind III digest with 10 units of this enzyme for 1 hour at 74 C. reaction assembly and primer digestion. PrimeSTAR HS DNA Polymerase with GC buffer provides reliable amplification, high accuracy, and high specificity in applications where amplification of high-gc DNA templates for cloning or library construction is required. 20 C Kit Components R044A PrimeSTAR HS DNA Polymerase (2.5 U/µL) 100 µl 2X PrimeSTAR GC Buffer (Mg 2+ )* 1.7 ml dntp Mixture (2.5 mm each) 800 µl * Mg 2+ concentration is 2 mm (2X) PrimeSTAR HS (Premix) PrimeSTAR HS DNA Polymerase Premix Cat.# R040A µl PrimeSTAR HS (Premix) is an optimized 2-fold mixture composed of PrimeSTAR HS DNA Polymerase, a high-fidelity DNA polymerase, reaction buffer and dntps. This is an excellent product for high-throughput applications because of the premixed reaction mixture, which saves time and reduces contamination risk. Kit Components R040A PrimeSTAR HS DNA Polymerase 1.25 units/25 µl dntp Mixture (2X conc.) ea. 0.4 mm PrimeSTAR Buffer (2X conc.) including 2 mm Mg 2+ Shipped and stored at 20 C. Avoid repeated freeze-thawing and vigorous mixing. Once thawed, dispense into PCR tubes and store at 20 C. 6

8 PrimeSTAR GXL DNA Polymerase PrimeSTAR GXL DNA Polymerase Cat.# R050A 250 Units PrimeSTAR GXL DNA Polymerase Cat.# R050B 1,000 Units Successful, Robust, High-Yield PCR regardless of Conditions Long PCR: Amplify products up to 30 kb (human genomic DNA), 40 kb (lambda DNA), or 13.5 kb (human cdna) Use on Standard and Challenging Templates alike: Outstanding performance on GC-rich or AT-rich templates and targets containing repeats Can be used with Samples Containing excess Nucleic Acid: Tolerates a wide range of template quantity, including high levels of template that inhibit other high-fidelity DNA polymerases Next Generation Sequencing (NGS) studies involving deep sequencing of the same region in many samples Features Highest Processivity: among commercially available high-fidelity DNA polymerases Antibody-Mediated Hot-Start Formulation Proven Performance: as reported in peer-reviewed literature Capable of outstanding performance for both routine high-fidelity PCR and challenging templates or reaction conditions, PrimeSTAR GXL DNA Polymerase is the most robust high-fidelity PCR enzyme commercially available. It provides high yield, high specificity, and high accuracy for not only standard reactions, but also excels in PCR with GC-rich templates, in the presence of excess template, and for amplification of PrimeSTAR Max DNA Polymerase Long products up to 30 kb (GXL). Simplify your PCR and save time by relying on one enzyme system works regardless of conditions, with minimal optimization required. PrimeSTAR GXL DNA Polymerase includes a modified PrimeSTAR HS enzyme and an additional elongation factor which in combination provide unsurpassed processivity. PrimeSTAR GXL DNA Polymerase has outstanding performance in reactions containing excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits PCR amplification by conventional polymerases because the amount of effective polymerase available is limited by nonspecific binding. The superior processivity of PrimeSTAR GXL DNA Polymerase prevents such inhibition by excess nucleic acid, resulting in a much higher success rate for PCR with minimal optimization of conditions required. Furthermore, the antibody-mediated hot start formulation prevents false initiation events during the reaction assembly due to mispriming and primer digestion. Kit Components R050A PrimeSTAR GXL DNA Polymerase (1.25 U/µL) 200 µl 2X PrimeSTAR GXL Buffer (Mg 2+ plus) 1.7 ml dntp Mixture (2.5 mm each) 800 µl * Mg 2+ concentration is 2 mm (2X) 20 C. Avoid repeated freeze-thaw cycles. PrimeSTAR MAX DNA Polymerase Cat.# R045A 100 Rxns PrimeSTAR MAX DNA Polymerase Cat.# R045B 400 Rxns Use Whenever Accuracy and Fidelity is Critical Cloning and Expression Studies Structure-Function Studies Analyses that involve Evolutionary Inferences (SNP analyses, evolutionary development experiments, etc.) Whenever Fast PCR Cycles are Needed, such as High-Throughput Studies Features Highest Fidelity: of any commercially available PCR polymerase Fastest Extension Speed: means less time required for PCR cycles Convenient Premix: assemble reactions in less time Antibody-Mediated Hot-Start Formulation Proven Performance: as reported in peer-reviewed literature PrimeSTAR Max DNA Polymerase is a unique high-performance DNA polymerase for PCR. PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extension speed of any commercially available enzyme, along with extremely high sensitivity, processivity, and specificity. It includes an elongation factor to provide efficient priming and extension, greatly reducing the time required for annealing and extension steps. As a result, PrimeSTAR Max DNA Polymerase can be used for exceptionally fast high-speed PCR. Since it is formulated as a premix that supports hot-start PCR, it's Outstanding Performance in All Conditions also excellent for high-throughput experiments. When you need fast reaction times and/or highly accurate amplification for cloning and expression, structural studies, or evolutionary analyses, PrimeSTAR Max DNA Polymerase is the enzyme of choice. The antibody-mediated hot start formulation prevents false initiation events during the reaction assembly due to mispriming and primer digestion. Moreover, it allows reaction assembly at room temperature. Since PrimeSTAR Max DNA Polymerase is configured as a 2-fold premix containing reaction buffer and dntp mixture, it allows rapid preparation of reactions and is useful for high-throughput applications. Kit Components R045A 2X PrimeSTAR Max Premix (Mg 2+ concentration: 2 mm) 625 µl x 4 20 C. Avoid repeated freeze-thaw cycles. When High Fidelity is Absolutely Critical PCR PRODUCTS 1 High Fidelity PCR Enzymes 7

9 1PCR PRODUCTS High Performance PCR TaKaRa Ex Taq TaKaRa Ex Taq Cat.# RR001A 250 U TaKaRa Ex Taq Cat.# RR001B 1000 U (4 250 U) TaKaRa Ex Taq Cat.# RR001C 3,000 U ( U) TaKaRa Ex Taq (Mg 2+ free Buffer) Cat.# RR01AM 250 U TaKaRa Ex Taq (Mg 2+ free Buffer) Cat.# RR01BM 1,000 U (4 250 U) TaKaRa Ex Taq (Mg 2+ free Buffer) Cat.# RR01CM 3,000 U ( U) High Yield and High Sensitivity PCR Improved Fidelity PCR Robust PCR Reproducible PCR Results Excellent on Difficult Templates Takara s Ex Taq combines the proven performance of Takara Taq with an efficient 3 g5 exonuclease activity for unsurpassed PCR performance. In routine PCR applications, the Ex Taq Polymerase and Ex Taq Buffer system gives higher yields and lower mutation rates (approximately 4.5X lower, as determined by the Kunkel method) than standard Taq DNA Polymerase. The system also allows amplification of longer products than Taq DNA Polymerase, with 20 kb lengths possible from genomic DNA and up to 30 kb possible from l DNA. Concentration 5 units/µl Form 20 mm Tris HCl (ph 8.0), 100 mm KCl, 0.1 mm EDTA, 1 mm DTT, 0.5% Tween 20, 0.5% Nonidet P-40, 50% Glycerol 20 C Purity Nicking activity, endonuclease and exonuclease activity are not detected after the incubation of 0.6 µg of supercoiled pbr322 DNA, 0.6 µg of λ DNA or 0.6 µg of λ-hind III digest with 10 units of this enzyme for 1 hour at 74 C. PCR Performance Test PCR performance by Polymerase Chain Reaction (PCR) is confirmed by using λ DNA as the template (amplified fragment: 20 kb). PCR performance is also confirmed via amplification of the β globin gene from a human DNA template. (amplified fragment : 17.5 kb). PCR Products Most PCR products amplified with TaKaRa Ex Taq have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. Also it is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Approximately 80% of the clones have a 3 A overhang. Kit Components RR001A TaKaRa Ex Taq 250 U (5 U/µL) 10X Ex Taq Buffer (contains 20 mm MgCl 2 ) 1 ml dntp Mixture (2.5 mm each dntp) 800 µl RR01AM TaKaRa Ex Taq 250 U (5 U/µL) 10X Ex Taq Buffer (without MgCl 2 ) 1 ml 20 mm MgCl 2 1 ml dntp Mixture (2.5 mm each dntp) 800 µl Amplification of a 6 kb Target from E. coli Genomic DNA with Takara Taq and Takara Ex Taq. PCR was performed using the indicated amounts of template, with either Takara Taq (Lanes T1-T4) or Takara Ex Taq (Lanes E1-E4). Each 50 µl reaction contained 1.25 units of enzyme. Lanes M contain l Hind III DNA Markers. 8

10 EmeraldAmp Max PCR Master Mix EmeraldAmp Max PCR Master Mix Cat.# RR320A 160 reactions (50 µl) EmeraldAmp Max PCR Master Mix Cat.# RR320B 800 reactions (50 µl) Used for Loading Samples Directly onto a Gel Immediately Following PCR Reaction Features High Yield: yields at least 10X more end product than Taq and can amplify targets up to 10 kb Excellent Performance: optimized buffer for better performance on GC or AT Rich Targets Convenient: just add template, primers and water to 2X Master Mix Eliminate Purification Steps: load PCR reaction directly onto a gel or use for TA cloning or direct sequencing without purification Emerald Green Loading Dye: track migration during electrophoresis Restriction Enzyme Digestion: digest PCR products directly in the PCR buffer Takara s EmeraldAmp Max PCR Master Mix includes an optimized buffer, PCR enzyme, dntp mixture, gel loading dye (green) and a density reagent in a 2X premix. Only primer and DNA template need to be added to easily complete the reaction. The mix can be used for routine PCR applications, and PCR reactions performed with this mix can be loaded directly onto a gel for electrophoresis. This product can amplify targets up to ~10kb (genomic DNA) and will work well on AT-rich or GC-rich templates. This premix can also yield at least 10X more end product than conventional PCR and has improved performance over standard Taq. The EmeraldAmp Max PCR Master Mix offers a powerful, flexible and convenient alternative for high yield, simple PCR applications. If 5 μl of the reaction mixture is used for electrophoresis with 1% Agarose L03 (Cat.# 5003), the blue dye marker is detected near 3~5 kb and the yellow is below 50 bp. Those dyes have absorptions at around 260nm and 420nm, respectively, so it is recommended to remove the dye by cutting out the gel or extracting DNA by NucleoSpin Extract II (Macherey-Nagel Cat.# /.250), for more applications. 20 C PCR Products Most PCR products amplified with EmeraldAmp Max PCR Master Mix have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. Also it is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Approximately 80% of the clones have a 3 A overhang. Kit Components RR320A (for 160 reactions, 50 µl each) EmeraldAmp Max PCR Master Mix (2X Premix) dh 2 O EmeraldAmp Max PCR Master Mix Company D Company E M M M M 4 1 ml 4 1 ml Template: Human Genomic DNA Target: 1. BCL2 581 bp (GC 67.7%) 2. IGFB bp (GC 72.3%) 3. jun 2025 bp (GC 65.2%) 4. IGF2R 3890 bp (GC 63.1%) PCR PRODUCTS 1 High Performance PCR Comparison of EmeraldAmp Max with Competitors for GC Rich Targets. Takara s EmeraldAmp Max PCR Master Mix was tested against Company D s and Company E s dye-added premixes. EmeraldAmp Max PCR Master Mix had greater reaction efficiency using GC rich targets than Company D s or Company E s dye-added premixes, resulting in significantly higher yields. In addition, EmeraldAmp Max PCR Master Mix provided greater specificity than Company D s dye-added premix. Premix Taq (Ex Taq Version) Premix Taq (Ex Taq Version) Cat.# RR003A 120 reactions (6 500 µl) Premix Taq is an optimized mixture composed of enzyme (TaKaRa Ex Taq ), reaction buffer and dntp mixture at a 2-fold concentration. Premix Taq (Ex Taq Version) is designed to allow quick and easy assembly of the reaction mixture. Kit Components RR003A Takara Ex Taq Premix* µl *Contains Takara Ex Taq, Ex Taq Buffer, 4 mm MgCl and dntps. 9

11 1PCR PRODUCTS High Performance PCR TaKaRa Ex Taq, Hot-Start Version TaKaRa Ex Taq Hot Start Version Cat.# RR006A 250 U TaKaRa Ex Taq Hot Start Version Cat.# RR006B 1000 U (4 250 U) Premix Ex Taq Hot Start Version Cat.# RR030A 100 reactions Robust, Specific Amplification with Reduced Background Amplifications Requiring Room-Temperature Reaction Assembly TaKaRa s Ex Taq HS includes a monoclonal antibody to Taq Polymerase which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. The antibody is then denatured in the initial PCR DNA-denaturation step, releasing the polymerase and allowing DNA synthesis to proceed. TaKaRa Ex Taq HS offers the same high performance as the standard TaKaRa Ex Taq including high yield, excellent sensitivity and fidelity 4.5X better than Taq Polymerase, along with the advantages of hot-start: lower background, increased specificity and room temperature reaction assembly. PCR Performance Enzyme performance is confirmed by successful amplification of a 20 kb l DNA template. Amplification of a single copy gene (b-globin) by PCR is also confirmed using human genomic DNA as the template (am plified frag ment: 17.5 kb). Inhibition of TaKaRa Ex Taq activity by the antibody is confirmed to be more than 90% after an incubation at 55 C for 10 min. Company A Takara Ex Taq M M M Amplification Efficiency Comparison Between TaKaRa s Ex Taq HS and a High-Grade Hot Start PCR Enzyme from Company A. A 7.5 kb human genomic DNA target was amplified from increasing amounts of template DNA. The results demonstrate the excellent sensitivity and yield of the TaKaRa Ex Taq HS. Lane M: l-hind III digest, Lane 1: 100 pg, Lane 2: 300 pg, Lane 3: 1 ng, Lane 4: 3 ng, Lane 5: 10 ng, Lane 6: 30 ng, Lane 7: 100 ng. PCR Products PCR products generated with TaKaRa Ex Taq HS contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency with T-vectors typically exceeds 80%. Kit Components RR006A TaKaRa Ex Taq HS 250 U (5 U/µL) 10X Ex Taq Buffer (contains 20 mm MgCl 2 ) 1 ml dntp Mixture (2.5 mm each dntp) 800 µl Use 1.25 U per 50 µl reaction. RR030A TaKaRa Ex Taq HS, Premix** µl ** Contains TaKaRa Ex Taq HS, 1.25 units/25 µl; dntp Mixture, 2X conc.; ea 0.4 mm Ex Taq Buffer, 2X conc.; including 4 nm Mg C. Avoid repeated freeze-thaws and vigorous stirring. Once thawed, aliquot into tubes and store at 20 C. Related Products Taq Antibody (Cat.# 9002A) Amplification of a 1.1 kb Bacillus sp. Genomic DNA Target. Lanes 1 and 2, TaKaRa Ex Taq HS; lanes 3 and 4, Company A; lanes 5 and 6, Company A with 10X Company A Buffer; lanes 7 and 8, Company C Polymerase; lanes 9 and 10, Company I Taq; lanes 11 and 12 Company B DNA Polymerase kb PerfectShot Ex Taq (Loading Dye Mix) PerfectShot Ex Taq (Loading Dye Mix) Cat.# RR005A 48 reactions (48 25 µl) Direct Loading of Sample on to a Gel after PCR Amplification PerfectShot Ex Taq is a convenient 2X PCR solution (supplied in a 1.2 ml PCR tube) which contains Takara s high performance Takara Ex Taq, reaction buffer, dntp mixture and loading dye (2-fold concentration). This premix allows direct loading of the amplified reaction mix onto a TAE gel without the addition of a separate gel loading buffer. Use of high yield Ex Taq plus the convenience of direct gel loading results in consistent, reproducible results for any application. Kit Components RR005A PerfectShot Ex Taq* 1.2 ml * Contains Ex Taq DNA Polymerase (1.25 U/25 µl), dntp (0.4 mm ea.), Ex Taq Buffer (2X), and Loading Dye (Orange G/Bromophenol Blue). 20 C. Thaw vials just prior to use. Aliquot to avoid repeated freeze-thaws. 10

12 EmeraldAmp Max HS PCR Master Mix EmeraldAmp Max HS PCR Master Mix Cat.# RR330A 160 reactions (50 µl) EmeraldAmp Max HS PCR Master Mix Cat.# RR330B 800 reactions (50 µl) Used for Loading Samples Directly onto a Gel Immediately following PCR Reaction Features Antibody Mediated Hot Start: minimize non-specific amplification and primer dimers High Yield: yields at least 10X more end product and can amplify targets up to 15 kb Excellent Performance: optimized buffer for better performance on GC-rich or AT-rich targets Convenient: just add template, primers and water to 2X Master Mix Eliminate Purification Steps: load PCR reaction directly onto a gel Emerald Green Loading Dye: track migration during electrophoresis Restriction Enzyme Digestion: digest PCR products directly in the PCR buffer Takara s EmeraldAmp Max HS PCR Master Mix includes a high yield, hot start PCR enzyme, optimized buffer, dntp mixture, gel loading dye (green) and a density reagent in a 2X premix. Only primer and DNA template need to be added to easily complete the reaction. The mix can be used for routine PCR applications, and PCR reactions performed with this mix can be loaded directly onto a gel for electrophoresis. This product can amplify targets up to ~15kb (genomic DNA) and will work well on high AT or GC templates. This premix can also yield at least 10X more end product than conventional PCR and has an improved performance over standard Taq. The EmeraldAmp Max HS PCR Master Mix offers a powerful, flexible and convenient alternative for high yield, simple PCR applications. If 5 μl of the reaction mixture is used for electrophoresis with 1% Agarose L03 (Cat.# 5003), the blue dye marker is detected near 3~5 kb and the yellow is below 50 bp. Those dyes have absorptions at around 260nm and 420nm, respectively, so it is recommended to remove the dye by cutting out the gel or extracting DNA by NucleoSpin Extract II (Macherey-Nagel Cat.# /.250) for more applications. Kit Components RR330A (for 160 reactions, 50 µl each) EmeraldAmp Max PCR Master Mix dh 2 O (2X Premix) 4 1 ml 4 1 ml PCR PRODUCTS 1 EmeraldAmp Max Company F Company F HS PCR Master Mix w/o dye w/ dye Company B Company A M M M M M M Template: Human Genomic DNA Target: 1. BCL2 581bp (GC 67.7%) 2. IGFB-1 987bp (GC 72.3%) 3. jun 2025bp (GC 65.2%) 4. IGF2R 3890bp (GC 63.1%) Template Amount: 50 ng Reaction Size: 25 µl High Performance PCR Comparison of EmeraldAmp Max HS with Competitors for GC Rich Targets. Takara s EmeraldAmp Max HS PCR Master Mix was tested against Company A s, Company B s and Company F s dye-added premixes, as well as Company F s premix without dye. EmeraldAmp Max HS PCR Master Mix showed the best reaction efficiency (with significantly higher yields compared to the other premixes) and was the only premix able to amplify the 3890 bp fragment. Taq Antibody Taq Antibody Cat.# 9002A 250 U Taq Antibody Cat.# 9002B 1000 U (4 250 U) Increasing Specificity and Lowering Background in PCR Reactions Room Temperature Reaction Assembly of PCR Reactions The Taq antibody is a monoclonal antibody to Taq Polymerase which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during PCR reaction assembly. The antibody is denatured in the initial PCR DNAdenaturation step, releasing the polymerase and allowing DNA synthesis to proceed. The addition of this antibody to Taq Polymerase, TaKaRa Ex Taq or TaKaRa LA Taq before a PCR reaction lowers background, increases specificity and allows room temperature reaction assembly. Concentration 5 U/µL Antibody Performance Inhibition of Taq polymerase activity by the antibody was confirmed to be more than 90% after incubation at 55 C for 10 min, after the incubation of the mixture of Taq Antibody and Taq Polymerase at 25 C for 10 min. Taq Polymerase activity was confirmed to recover almost 100% activity in a reaction at 55 C for 10 minutes after heat treatment at 94 C for 30 seconds. 20 C 11

13 1PCR PRODUCTS TaKaRa LA Taq TaKaRa LA Taq (without Mg 2+ ) Cat.# RR002A 125 U TaKaRa LA Taq Cat.# RR002M 250 U TaKaRa LA Taq DNA Polymerase Cat.# RR002B 1,000 U (4 250 U) TaKaRa LA Taq DNA Polymerase Cat.# RR002C 3,000 U ( U) Robust Amplification of Long DNA Templates (up to 48 kb possible) with Minimal Optimization Longer and More Accurate Amplification of Genomic PCR Products TaKaRa LA Taq is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using TaKaRa LA Taq, routine extension to 20 kb and up to 48 kb is possible (depending on template type) with less optimization than other long PCR systems. Because of the presence of the proofreading polymerase, the fidelity is significantly better (6.5X) than that of Taq Polymerase alone. Kit Components RR002M TaKaRa LA Taq 250 U (5 U/µL)* 10X LA PCR Buffer II (contains 25 mm MgCl 2 ) 1 ml dntp Mixture (2.5 mm each dntp) 800 µl RR002A TaKaRa LA Taq 125 U (5 U/µL)* 10X LA PCR Buffer II (without Mg 2+ ) 1 ml 25 mm MgCl 2 1 ml dntp Mixture (2.5 mm each dntp) 400 µl * Use 2.5 U per 50 µl reaction. PCR Products PCR products generated with TaKaRa LA Taq contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency of products <5 kb into T vectors exceeds 80%. Cloning efficiency of >5 kb products into T vectors decreases with product size. PCR Performance Enzyme performance is confirmed by successful amplification of a 35 kb l DNA template and a 17.5 kb human genomic DNA template. 20 C. Avoid repeated freeze-thaws of TaKaRa LA Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. High Performance PCR M M2 M1: phy Marker 1: 0.5 kb 2: 1 kb 3: 2 kb 4: 4 kb 5: 6 kb 6: 8 kb 7: 10 kb 8: 12 kb 9: 15 kb 10: 20 kb 11: 28 kb 12: 35 kb M2: λ-hind III digest Amplification of l DNA Fragments from kb in Size (different primer sets) using TaKaRa LA Taq. TaKaRa LA Taq was used to amplify the various fragments and generated high product yields, even with very long (28 kb) fragments. One-Shot LA PCR Mix One-Shot LA PCR Mix Cat.# RR tubes (25 µl ea.) Convenient, Reliable Amplification of Long DNA Fragments Longer and More Accurate Genomic PCR Amplifications Increased Reproducibility and Minimal Optimization for Long PCR Excellent for High-Throughput Studies The One-Shot LA PCR Mix is an optimized mixture composed of TaKaRa LA Taq, Buffer and dntps at a 2X concentration in 25 µl aliquots. These aliquots are predispensed into 0.2 ml PCR tubes. By addition of an equal volume of solution containing the template and primers to each tube, a complete, ready-to-amplify reaction is obtained. Kit Components RR004 One-Shot LA PCR Mix* µl *Contains TaKaRa LA Taq, LA PCR Buffer II (2X conc.; including 5 mm Mg 2+ ) and dntps. PCR Products PCR products generated with TaKaRa LA Taq contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency of products <5 kb into T vectors exceeds 80%. Cloning efficiency of >5 kb products into T vectors decreases with product size. 20 C 12

14 LA PCR Kit Version 2.1 LA PCR Kit Version 2.1 Cat.# RR013A 50 reactions LA PCR Kit Version 2.1 Cat.# RR013B 100 reactions Amplification of Large DNA Templates (30 kb genomic or 48 kb l DNA) Longer and More Accurate Genomic PCR Products Includes GC buffers at 2X Concentration for Templates of High GC-Content with Strong Secondary Structure The LA PCR Kit, Ver. 2.1 includes all the reagents necessary for amplification of long DNA templates with routine extension to 30 kb (up to 48 kb is possible with some templates.) The Version 2.1 kit combines TaKaRa LA Taq with an optimized buffer system resulting in longer and more accurate PCR products than conventional PCR reagents. High fidelity (6.5-fold better than conventional Taq DNA Polymerase) is facilitated by an efficient 3 g5 exonuclease activity. The Version 2.1 kit contains a control template and primers to ensure premium PCR performance. Amplification of Human Genomic DNA with TaKaRa LA Taq. Purified human genomic DNA (500 ng in a 50 µl reaction) was used as template for PCR with TaKaRa LA Taq. The primer sets used amplified target regions in the b-globin gene cluster and the TPA gene. The sizes of the amplified products were 17.5 kb (lane 1), 21.5 kb (lane 2), and 27 kb (lane 3). Lane M contains High MW Markers (Life Technologies). Includes Everything Needed for Long PCR Optimization M kb kb kb Kit Components RR013A TaKaRa LA Taq 125 U (5 U/µL) 10X LA PCR Buffer II (contains 25 mm Mg 2+ ) 250 µl 10X LA PCR Buffer II (without Mg 2+ ) 250 µl MgCl 2 (25 mm) 500 µl dntp Mixture (2.5 mm each dntp) 400 µl Control Template (100 ng/µl HT29 DNA) 10 µl Control Primer LA3 (10 pmol/µl)* 10 µl Control Primer LA4 (10 pmol/µl)* 10 µl λ-hind III MW Markers (100 ng/µl) 20 µl 2X GC Buffer I (contains 5 mm MgCl 2 ) 1.25 ml 2X GC Buffer II (contains 5 mm MgCl 2 ) 1.25 ml Control Primer GC1 (10 pmol/µl)** 10 µl Control Primer GC2 (10 pmol/µl)** 10 µl * Amplifies a 17.5 kb region of the Control Template. ** Amplifies a 1,255 bp GC-rich region of the Control Template. PCR Products PCR products generated with TaKaRa LA Taq contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency of products <5 kb into T vectors exceeds 80%. Cloning efficiency of >5 kb products into T vectors decreases with product size. 20 C PCR PRODUCTS 1 High Performance PCR 13

15 1PCR PRODUCTS TaKaRa LA Taq Hot-Start Version TaKaRa LA Taq Hot Start Version Cat.# RR042A 125 U TaKaRa LA Taq Hot Start Version Cat.# RR042B 500 U (4 125 U) Long Amplifications Synthesizing Products up to 30 kb (genomic DNA) and 48 kb (l DNA) High Fidelity Amplifications (6.5X higher than Taq DNA Polymerase) Enzyme-Buffer System Allows Robust Amplification and High Yield with Less Need for Optimization Reduced Background and Increased Reaction Specificity Compatible with Multiplex PCR TaKaRa LA Taq Hot-Start consists of TaKaRa LA Taq plus a monoclonal antibody to Taq Polymerase. It retains all of the high performance features of TaKaRa LA Taq and provides increased reaction specificity and reduced background. The key features of TaKaRa LA Taq are: synthesis of products up to 30 kb (genomic DNA) and 48 kb (l DNA), 6.5X higher fidelity than Taq DNA Polymerase, and less optimization and greater product yield than other long polymerases due to the robust enzyme-buffer system. Room temperature reaction assembly is possible with this formulation. Kit Components RR042A TaKaRa LA Taq HS 125 U (5 U/µL)* 10X LA PCR Buffer II (Mg 2+ ) 25 mm dntp Mixture (2.5 mm each dntp) 400 µl * Use 2.5 U per 50 µl reaction. PCR Products PCR products generated with TaKaRa LA Taq contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency of products <5 kb into T vectors exceeds 80%. Cloning efficiency of >5 kb products into T vectors decreases with product size. PCR Performance Performance is confirmed by successful amplification of a 35 kb l DNA template and a 17.5 kb human genomic DNA. Inhibition of TaKaRa LA Taq activity by the anti-taq antibody (hot-start formulation) is confirmed to be more than 90% after an incubation of 55 C for 10 min. 20 C. Avoid repeated freeze-thaws of TaKaRa LA Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. High Performance PCR LA PCR Genome DNA Set LA PCR Genome DNA Set Cat.# reactions Human Genomic and E. coli Genomic DNA Control Templates and Primers for Use with the LA PCR Kit This set includes highly purified, high molecular weight human genomic DNA and E. coli genomic DNA, along with appropriate primers. These templates and primers are intended for use as controls for the LA PCR Kit and are useful for optimizing PCR conditions for a variety of templates. Kit Components 9060 Human genomic DNA 10 µg E. coli genomic DNA 2 µg Human-1 Primer* 200 pmol Human-2 Primer* 200 pmol E. coli-1 Primer** 200 pmol E. coli-2 Primer** 200 pmol * Amplimer size: 17.5 kb ** Amplimer size: 20 kb DNA: 4 C (Can be stored at 20 C. Avoid freeze-thaw cycles.) Primers: 20 C 14

16 TaKaRa LA Taq with GC Buffers TaKaRa LA Taq with GC Buffers Cat.# RR02AG 125 U Amplification of Large DNA Templates (up to 48 kb possible) Longer and More Accurate Genomic PCR Amplification Amplification of GC-rich Templates TaKaRa LA Taq is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using TaKaRa LA Taq, routine extension to 20 kb is possible (up to 48 kb on some templates), with less optimization than other long PCR systems. Because of the presence of the proofreading polymerase, fidelity is significantly better (6.5X) than Taq Polymerase alone. The GC-optimized Buffers I and II are specifically designed for DNA templates with high GC content or a significant amount of secondary structure. GC Buffer I is recommended for amplification of fragments $5kb. GC Buffer II is recommended for 2 3 kb fragments with up to 73% GC content. Kit Components RR02AG TaKaRa LA Taq 125 U (5 U/µL)* 2X GC Buffer I (contains 5 mm MgCl 2 ) 1.25 ml 2X GC Buffer II (contains 5 mm MgCl 2 ) 1.25 ml dntp Mixture (2.5 mm each dntp) 400 µl * Use 2.5 U per 50 µl reaction. PCR Products PCR products generated with TaKaRa LA Taq contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency of products <5 kb into T vectors exceeds 80%. Cloning efficiency of >5 kb products into T vectors decreases with product size. PCR Performance Performance is confirmed by successful amplification of a 35 kb l DNA template, and a 17.5 kb human genomic DNA with GC Buffer I. Amplification of the c jun proto oncogene (1,255 bp, 65% GC content) by PCR is also confirmed using human genomic DNA as the template with GC Buffer I and GC Buffer II. 20 C. Avoid repeated freeze-thaws of TaKaRa LA Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. PCR PRODUCTS 1 M M2-2.1 kb Comparison of Amplification Efficiency between TaKaRa LA Taq with GC Buffer and TaKaRa LA Taq using a GC-rich Target Fragment. A 2.1 kb mouse rrna gene with 63.4% GC was amplified from 1 ng of mouse genomic DNA. The results show TaKaRa LA Taq with GC Buffer gave optimal results in amplification of the 2.1 kb fragment with both excellent yields and high specificity. M1.: l -Hind III digest 1 & 2: LA Taq / LA PCR Buffer II 3 & 4: LA Taq with GC Buffer/ GC Buffer I 5 & 6: LA Taq with GC Buffer/ GC Buffer II M2: phy Marker M kb -262 kb Amplification of a Huntington s Disease Gene (high GC content). Purified human genomic DNA (100 ng in a 50 µl reaction) was used as a template for PCR with TaKaRa LA Taq and either LA PCR Buffer II (lane 1), GC Buffer I (lane 2), or GC Buffer II (lane 3), or with a competing DNA Polymerase and high GC Kit (lane 4). The primers amplified regions of the HD gene IT15 CAG repeat. The sizes of the amplified products were 262 bp (GC content 73%), and 358 bp (GC content 71.5%). Lane M contains a 100 bp ladder. High Performance PCR 15

17 1PCR PRODUCTS High Speed PCR SapphireAmp Fast PCR Master Mix SapphireAmp Fast PCR Master Mix Cat.# RR350A 160 reactions (50 µl) SapphireAmp Fast PCR Master Mix Cat.# RR350B 800 reactions (50 µl) High Speed Amplification with the Convenience of an Added Dye for Direct Loading on a Gel Features Faster than Standard Taq: reactions completed in 1/2 the time of conventional Taq Restriction Enzyme Digestion can be Directly Performed on PCR Products: in the PCR buffer PCR Reaction can be Directly Loaded onto a Gel: without purification or addition of other reagents Saves Time, Convenient and Eliminates Purification Steps: with no reduction in yield Excellent for High-Throughput Studies SapphireAmp Fast PCR mix contains a hot start PCR enzyme, optimized buffer, dntp mixture, gel loading dye (blue) and a density reagent in 2X premix. PCR reactions with this mix can be completed in half the time required for conventional Taq amplifications. Amplification of human genomic DNA targets 2 kb in length can be completed in 1 hour, and amplifications of human genomic DNA targets up to 6 kb are possible using SapphireAmp. This product is well-suited for E.coli-based colony PCR to assess presence of inserts of up to ~5 kb, and colony checks can be completed in about 1 hour. TaKaRa SapphireAmp Fast Company A Company B SapphireAmp reactions can be assembled by simply adding primer and DNA template to the premix. Reactions performed with this mix can be loaded directly onto a gel for electrophoresis. Restriction digestion of PCR products is possible in SapphireAmp reaction buffer. SapphireAmp offers a powerful, fast, flexible and convenient alternative for both simple and complex PCR applications. 20 C (at 4 C for 3 months) M M M M PCR Products Most PCR products amplified with SapphireAmp have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. Also it is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Kit Components RR350A SapphireAmp Fast PCR Master Mix (2X Premix) dh 2 0 Fast PCR Template: Human Genome 100 ng/50 ul Targets: 1: p kb (54%) 2: FFAR2 1.0 kb (59%) 3: DCLRE1A 2.0 kb (38%) 4: p kb (48%) 5: IGF2R 5.9 kb (50%) 4 1 ml 4 1 ml Comparison of SapphireAmp Fast to Competitors with Standard Targets Takara s SapphireAmp Fast PCR Master Mix was tested against Company A s and Company B s dye-added premixes. SapphireAmp Fast PCR Master Mix provided similar yields of DNA up to 4.2 kb compared to Company A s and Company B s dye-added premixes in less time. In addition, SapphireAmp Fast PCR Master Mix was able to amplify 4.2 and 5.9 kb fragments with significant yields vs Company A and Company B. 16

18 SpeedSTAR HS DNA Polymerase SpeedSTAR HS DNA Polymerase Cat.# RR070A 250 U SpeedSTAR HS DNA Polymerase Cat.# RR070B 1000 U (4 250 U) High Speed Amplification Reduces Reaction Time by Two-Thirds Robust Amplification of Large DNA Templates Features High Speed Amplification: Amplify a 2 kb fragment in as little as 30 minutes Excellent Efficiency: Robust performance, comparable to high yield polymerases Long Fragments: Optimized two-buffer system allows amplification of fragments up to 20 kb with reduced optimization No Special Instrument Needed: Reduce reaction times by two-thirds without purchasing a specialized instrument SpeedSTAR HS DNA Polymerase is a convenient, efficient DNA polymerase specially optimized for fast PCR. Extension times of as little as 10 sec/kb are possible (compared to 60 sec/kb with general enzymes), dramatically reducing total reaction times. SpeedSTAR reactions can be performed using standard PCR instrumentation, eliminating the requirement for special equipment. The robust, two-buffer system facilitates efficient amplification of varying size fragments (up to 20 kb) with less optimization than other polymerases. In addition, the hot-start formulation is antibody mediated and provides convenience and reduced background. TaKaRa Z-Taq TaKaRa Z-Taq Cat.# R006A 200 U TaKaRa Z-Taq Cat.# R006B 800 U (4 200 U) Rapid PCR Amplification of Longer PCR Products TaKaRa Z-Taq is a novel enzyme that possesses superior properties compared to conventional Taq DNA polymerase. TaKaRa Z-Taq is characterized by 5-fold greater processivity than Taq DNA Polymerase, resulting in rapid amplification times in PCR applications. Using TaKaRa Z-Taq, a 1 kb DNA product can be amplified from genomic DNA in 20 min. total time using a 30-cycle, two-step amplification protocol. The improved processivity of TaKaRa Z-Taq also enables the amplification of longer templates than Taq DNA Polymerase. For example, human genomic DNA templates up to 17.5 kb and l DNA templates of 20 kb have been successfully amplified. Kit Components RR070A SpeedSTAR HS DNA Polymerase (5 units/µl) 50 µl 10X Fast Buffer I (Mg 2+ *) 1 ml 10X Fast Buffer II (Mg 2+ *) 1 ml dntp Mixture (ea. 2.5 mm) 800 µl *Mg 2+ Concentration: 10X Fast Buffer I, 30 mm; 10X Fast Buffer II, 20 mm Purity Nicking activity, endonuclease and exonuclease activity are not detected after the incubation of 0.6 µg of supercoiled pbr322, 0.6 µg of l DNA or 0.6 µg of l-hind III digest with 10 units of this enzyme for 1 hour at 74 C. PCR Products Most PCR products amplified with SpeedSTAR HS DNA Polymerase have one A added at 3 -terminus. PCR products can be directly used for cloning into a T-vector. It is also possible to clone the product into blunt-ended vectors after blunting and phosphorylation. 20 C Kit Components R006A TaKaRa Z-Taq 200 U (2.5 U/µL)* 10X Z-Taq Buffer (with 30 mm MgCl 2 ) 800 µl dntp Mixture (2.5 mm each dntp) 800µL *Use 1.25 U per 50 µl reaction. PCR Products PCR products generated with TaKaRa Z-Taq contain a mixture of 3 -A overhangs and blunt ends which allows >80% cloning efficiency in T-vectors. PCR Performance Performance of rapid DNA amplification by PCR is confirmed by using a 10 kb l DNA tem plate. 20 C. Avoid repeated freeze-thaws of TaKaRa Z-Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. PCR PRODUCTS 1 High Speed PCR 17

19 1PCR PRODUCTS General PCR Products TaKaRa Taq and Premix Taq (TaKaRa Taq Version) TaKaRa Taq Cat.# R001A 250 U TaKaRa Taq Cat.# R001B 1000 U (4 250 U) TaKaRa Taq Cat.# R001C 3000 U ( U) TaKaRa Taq (Mg 2+ free buffer) Cat.# R001AM* 250 U TaKaRa Taq (Mg 2+ free buffer) Cat.# R001BM* 1000 U (4 250 U) TaKaRa Taq (Mg 2+ free buffer) Cat.# R001CM* 3000 U ( U Premix Taq (TaKaRa Taq Version) Cat.# R004A 120 reactions (6 500 µl) *Contains magnesium-free buffer and a separate tube of 25 mm MgCl 2. Routine PCR Thermal Cycle Sequencing TaKaRa Taq is a highly purified, recombinant, thermostable, 94 kda DNA polymerase encoded by the DNA polymerase gene of the Thermus aquaticus YT-1 strain which has been cloned into Escherichia coli. It has been shown to have essentially the same characteristics as native Taq DNA polymerase. TaKaRa Taq is a versatile thermostable DNA polymerase suitable for a variety of standard PCR applications. The enzyme is also supplied in a premix format (a mixture of enzyme, buffer, and dntps) which simplifies the setup of PCR reactions and minimizes pipetting steps. Kit Components R001A TaKaRa Taq 250 U (5 U/µL)* 10X PCR Buffer (contains 15 mm MgCl 2 ) 1 ml dntp Mixture (2.5 mm each dntp) 800 µl R001AM TaKaRa Taq 250 U (5 U/µL)* 10X PCR Buffer (without Mg 2+ ) 1 ml 25 mm MgCl 2 1 ml dntp Mixture (2.5 mm each dntp) 800 µl R004A 2X Taq Polymerase Premix** µl * Use 1.25 U per 50 µl reaction. ** Contains TaKaRa Taq, PCR Buffer and dntps. PCR Performance Enzyme performance is confirmed by successful amplification of an 8 kb l DNA tem plate. Amplification of a single copy gene by PCR is also confirmed using a 2.9 kb human genomic DNA template and human p53-primers. Source Recombinant E. coli. Contamination with E. coli DNA is below 10 fg as shown by nested PCR. 20 C. Avoid repeated freeze-thaws of TaKaRa Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. M M 1 2 Amplification of l DNA. A sample containing 1 ng of l DNA was amplified with TaKaRa Taq, using various sets of primers. The PCR products were analyzed by agarose gel electrophoresis: lane 1, 4 kb; lane 2, 6 kb; lane 3, 8 kb; lane 4, 10 kb; lane M, l-hind III DNA Markers. Amplification of a Single-Copy Gene. Amplification was performed using TaKaRa Taq, human placentai genomic DNA (100 ng) as a template, and primers that amplified two different regions of the human p53 gene. The size of the amplified products were 1.2 kb (lane 1) and 2.9 kb (lane 2). Lane M contains l-hind III DNA Markers. 18

20 EmeraldAmp GT PCR Master Mix EmeraldAmp GT PCR Master Mix Cat.# RR310A 160 reactions (50 µl) EmeraldAmp GT PCR Master Mix Cat.# RR310B 800 reactions (50 µl) Used for Loading Samples Directly onto a Gel Immediately Following PCR Reaction Features Improved Performance: optimized buffer for better performance on GC or AT Rich Targets Convenient: just add template, primers and water to the 2X Master Mix Eliminate Purification Steps: load the PCR reaction directly onto a gel or use for TA cloning or direct sequencing without purification Emerald Green Loading Dye: allows tracking during electrophoresis Restriction Enzyme Digestion: digest PCR products directly in the PCR buffer Low Contamination Grade Taq: <10 fg of E. coli DNA is present in a standard aliquot containing 1 unit of Taq Takara s EmeraldAmp GT PCR for great performance in standard PCR applications is a PCR Master Mix including an optimized buffer, PCR enzyme, dntp mixture, gel loading dye (green) and a density reagent in 2X premix. The EmeraldAmp GT PCR Master Mix reaction assembly can be performed by simply adding primer and DNA template to the tube of premix. Reactions performed with these mixes can be loaded directly onto an agarose gel for electrophoresis, and restriction digestion of PCR products is possible in the EmeraldAmp GT reaction buffer. This product can be used for PCR products up to ~5kb of genomic DNA and will work with templates with over 50% GC and AT content. The EmeraldAmp GT PCR Master Mix offers a powerful, flexible and convenient reagent for simple PCR applications. If 5 μl of the reaction mixture is used for electrophoresis with 1% Agarose L03 (Cat.# 5003), the blue dye marker is detected near 3~5 kb and the yellow is below 50 bp. Those dyes have absorptions at around 260nm and 420nm, respectively, so it is recommended to remove the dye by cutting out the gel or extracting DNA by NucleoSpin Extract II. (Macherey-Nagel Cat.# /.250) Kit Components R310A (for 160 reactions, 50 µl each) EmeraldAmp GT PCR Master Mix dh 2 O 20 C EmeraldAmp GT PCR Master Mix Company B Company C M M M M (2X Premix) 4 1 ml 4 1 ml PCR Products Most PCR products amplified with EmeraldAmp GT PCR Master Mix have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. It is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Approximately 80% of the clones have a 3 A overhang. Template: Human Genomic DNA Target: 1. BCL2 581bp (GC 67.7%) 2. IGFB-1 987bp (GC 72.3%) 3. jun 2025bp (GC 65.2%) 4. IGF2R 3890bp (GC 63.1%) PCR PRODUCTS 1 General PCR Products Comparison of EmeraldAmp GT with Competitors for GC Rich Targets. Takara s EmeraldAmp GT PCR Master Mix was tested against Company B s and Company C s dye-added premixes. EmeraldAmp GT PCR Master Mix provided greater reaction efficiency than Company B s and C s dye-added premixes with GC-rich human DNA templates. Takara s EmeraldAmp GT PCR Master Mix provided excellent yield with GC-rich DNA fragments up to 2025 bp. PCR Amplification Kit PCR Amplification Kit Cat.# R reactions Amplification of DNA from any Template via PCR The PCR Amplification Kit is designed to perform PCR on any DNA tem plate and supplies all the reagents necessary for PCR, including dntps. The kit also includes a control template and primers for verifying am pli fi ca tion conditions, a Taq dilution buffer, and size markers. 20 C. Avoid repeated freeze-thaws of TaKaRa Taq and dntps. Once thawed, aliquot into separate tubes and store at 20 C. Great PCR Optimization Kit Components R011 TaKaRa Taq (5 U/µL) 250 U dntp Mixture (2.5 mm each) 1.28 ml 10X PCR Buffer (contains 15 mm MgCl 2 ) 1 ml 10X PCR Buffer (without Mg 2+ ) 1 ml MgCl 2 (25 mm) 1 ml Control Template (1 µg/ml l DNA) 100 µl Control Primer 1 (20 pmol/µl) 50 µl Control Primer 2 (20 pmol/µl) 50 µl Control Primer 3 (20 pmol/µl) 50 µl λ EcoT 14 I Digest (100 ng/µl) 40 µl 6X Loading Buffer 1 ml (36% Glycerol, 0.05% Bromophenol Blue, 30mM EDTA, 0.05% Xylene cyanol) 19

21 1PCR PRODUCTS TaKaRa Taq Hot-Start Version TaKaRa Taq Hot-Start Version Cat.# R007A 250 U TaKaRa Taq Hot-Start Version Cat.# R007B 1000 U (4 250 U) TaKaRa Taq Hot-Start Version (premix) Cat.# R028A 100 reactions (50 µl rxn) Multiplex PCR Reduced Background and Increased Reaction Specificity Reliable, Consistent Standard and qpcr Amplifications Features Increased Specificity: in Standard Amplification High Throughput Capacity: in Standard PCR Room Temperature Assembly Excellent Multiplex PCR TaKaRa Taq HS contains a mixture of Taq Polymerase and a monoclonal antibody to Taq Polymerase, which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. The antibody is then denatured in the initial PCR DNA-denaturation step, releasing the polymerase and allowing DNA synthesis to proceed. Premix Taq Hot Start Version TaKaRa Taq HS, Premix is an optimized mixture (2-fold concentration) of TaKaRa Taq HS, a reaction buffer and dntps. Kit Components R007A TaKaRa Taq HS 250 U (5 U/µL)* 10X PCR Buffer (contains 15 mm MgCl 2 ) 1 ml dntp Mixture (2.5 mm each dntp) 800 µl * Use 1.25 U per 50 µl reaction. R028A TaKaRa Taq HS, Premix* µl * Contains dntp Mixture, TaKaRa Taq HS (1.25 U/25 μl) 2X conc; ea. 0.4 mm and PCR buffer 20 mm Tris-HCl, ph 8.3, 100 mm KCl and 3 mm 2X conc. MgCl 2 20 C. Avoid repeated freeze-thaw cycles. Aliquot into separate tubes and store at 20 C. General PCR Products Application: For Multiplex PCR using TaKaRa Taq Hot Start Materials and Methods PCR reactions were performed using TaKaRa Taq or TaKaRa Taq HS to amplify a human genomic DNA template with eight different primer pairs, each specific for a target ranging from 84 to 432 bp in size. Lanes 1 8 contain individual reactions for each primer pair amplified using TaKaRa Taq HS. Lanes 9 and 10 include multiplex PCR reactions containing all eight primer pairs in a single tube, amplified with either Taq (lane 9) or TaKaRa Taq HS (lane 10). The individual reactions were cycled under the following conditions: 94 C, 30 sec 1 cycle i 94 C, 30 sec 55 C, 30 sec 30 cycles 72 C, 60 sec Results The results show that multiplex PCR using TaKaRa Taq HS results in target amplification efficiencies equivalent to that of separate (single target) amplification reactions. In addition, TaKaRa Taq HS demonstrates superior efficiency and specificity over standard Taq Polymerase in this multiplex PCR application. M M1 The multiplex reactions were cycled under the following conditions: 94 C, 30 sec 1 cycle i 94 C, 30 sec 57 C, 30 sec 30 cycles 72 C, 60 sec i 72 C, 90 sec Excellent for Multiplex PCR Amplification of Various Human Genomic DNA Fragments using a Standard Taq DNA Polymerase and TaKaRa Taq Hot-Start DNA Polymerase. PCR reactions were performed using human genomic DNA as a template and 8 different primer pairs for each single fragment. All fragments are amplified in a multiplex reaction, Lane 9, amplified using Standard Taq. Lane 10, amplified using TaKaRa Taq Hot-Start. 20

22 EpiScope MSP Kit EpiScope MSP Kit Cat.# R100A 200 reactions EpiScope MSP Kit Cat.# R100B 400 reactions EpiScope Methylated HeLa Genomic DNA Cat.# µg Methylation Specific PCR for DNA Methylation Analysis Features High Performance: Superior amplification efficiency and specificity Robust: Differentiate methylated/unmethylated DNA with just a single CpG site in the primers Optimized: For real-time monitoring using SYBR Green Versatile: Perform both real-time and endpoint PCR using the same PCR conditions EpiScope MSP Kit contains PCR reagents designed exclusively for methylationspecific PCR (MSP). A specific enzyme combined with an optimized buffer allows MSP analyses using a bisulfite-treated DNA template containing uracil. This kit provides a higher amplification efficiency and greatly improved ability to differentiate methylated/unmethylated DNA compared with traditional PCR reagents. The reaction system has been optimized for real-time monitoring using SYBR Green I 1, and makes it possible to perform both real-time PCR and endpoint detection under the same reaction conditions. A positive control, EpiScope Methylated HeLa gdna (Cat.# 3520), is sold separately. This genomic DNA, which was purified from human HeLa cells, has been highly methylated using CpG methylase. It can be used as a positive control in MSP, Bisulfite Sequencing, Combined Bisulfite Restriction Analysis (COBRA), Methylated CpG Island Recovery Assay (MIRA), and other DNA methylation analysis methods. 1 TAKARA BIO is under a license agreement with Molecular Probes Inc. for the use of SYBR Green I as a reagent for research purposes. SYBR is a registered trademark of Molecular Probes Inc. DNA Fragmentation Kit Principle of MSP The first step is to identify a nucleotide region whose sequence is subject to change by bisulfite treatment depending on the methylation status of the CpG sequence. Next, design two primers for this region; one for methylated CpG DNA (M primer), and the other for unmethylated DNA (UM primer). The last step is PCR amplification. Kit Components R100A 2X MSP Buffer (Mg 2+ plus, dntp plus) *1 5 X 1 ml MSP Enzyme 240 µl 100X SYBR Green I 100 µl ROX Reference Dye (50 conc.) *2 200 µl ROX Reference Dye II (50 conc.) *2 200 µl *1 The Mg 2+ concentration (2X ) is 4 mm, and the dntp concentration (2X ) is 400 µm). *2 This component is to be used for analyses using a device that corrects fluorescent signals between wells, such as the real-time PCR device by Applied Biosystems. Please use ROX Reference Dye for Applied Biosystems StepOnePlus Real-Time PCR System or ROX Reference Dye II for 7500 and 7500 Fast Real-Time PCR System. This component is not required when using a real-time PCR device such as Thermal Cycler Dice Real Time System II (Cat.# TP900, not available in the US and Europe) or an ordinary PCR device for electrophoretic analysis Form: 10mM Tris-HCI (ph8.0) 1mM EDTA 20 C* * Protect 100X SYBR Green I from light. Note: The reaction composition and the PCR condition are the same for real-time PCR and endpoint detection. Even for endpoint detections, be sure to add 100X SYBR Green I. DNA Fragmentation Kit Cat.# reactions Perform random fragmentation of genomic DNA and other longchain dsdnas. Pretreatment of methylated DNA. This kit is designed to perform random fragmentation of genomic DNAs and other long-chain dsdnas by enzyme treatment without any special apparatus such as a sonicator, and then to blunt the obtained DNA fragment. Blunt-end fragments may be inserted into blunt-end vectors. The reaction may be stopped after fragmentation is complete if no blunting is required. This kit may also be used to pre-treat samples in methylated DNA and to prepare samples for high speed sequence analysis. Use for Pre-treatment of Methylated DNA! Kit Components 6137 Enzyme 1 20 µl Dilution Buffer- 1 1,040 µl A Solution 20 µl B Solution 50 µl Stop Solution 400 µl 150 mm MgCl 2 40 µl Dilution Buffer µl Enzyme µl 0.5 M EDTA 50 µl dh 2 O 10 X 1 ml Materials Required but not Provided Reagents: Electrophoresis buffer. We recommend using a loading buffer that contains a dye (e.g., Orange G) that would not overlap on the electrophoresis gel with bands for fragments that are 100 1,000 bp. Bromophenol blue or xylene-cyanol do overlap with DNA fragments this size. Apparatus: Thermal Cycler (at least 1; 2 are preferred) PCR PRODUCTS 1 General PCR Products 21

23 1PCR PRODUCTS Deoxynucleotide Triphosphates (dntps) datp Cat.# µmol dctp Cat.# µmol dgtp Cat.# µmol dttp Cat.# µmol dntp Mixture Cat.# µmol ea./1.28 ml at 2.5 mm conc. ea dntp Set Cat.# µmol ea./4 tubes Amplification of DNA (PCR) Reverse Transcription and RT-PCR Synthesis of cdna Common Molecular Biology Procedures such as Cloning, Sequencing and Labeling of DNA The purity and quality of deoxynucleotide triphosphates (dntps) are vital to the success of demanding applications such as PCR and RT-PCR. These dntps are $98% pure and are quality-tested in a variety of applications. Individual dntps are supplied in solution at a concentration of 100 mm, and can be diluted with water or buffer as needed. The dntp mixture from Takara is a premixed, ready-to-use solution containing each dntp at a concentration of 2.5 mm. This preparation offers added convenience, minimizes pipetting steps and can be added directly to amplification reactions. Form Aqueous solution (sodium salts) ph C. Avoid repeated freeze-thaws. Once thawed, aliquot into separate tubes and store at 20 C. Excellent Purity and Quality dntp s General PCR Products RT-PCR Products Reverse Transcription PCR PrimeScript 1st Strand cdna Synthesis Kit PrimeScript RT-PCR Kit PrimeScript One Step RT-PCR Kit PrimeScript RT Reagent Kit for Real Time High fidelity PrimeScript RT-PCR Kit 22

24 PrimeScript RT-PCR Kit PrimeScript RTase Cat.# RR014A 50 reactions PrimeScript RTase Cat.# RR014B 200 reactions Robust Reverse Transcription and Amplification of Full-Length cdnas even from Challenging RNA Templates Features Complete Elongation: PrimeScript RTase works efficiently on higher structured RNA templates. High Yield of Full-Length cdna: PrimeScript RTase offers high yield transcription of full length cdnas of up to 12 kb High Specificity, Efficient Elongation: RT-PCR kits combining PrimeScript RTase and TaKaRa Ex Taq HS minimizing false priming and allowing robust amplification from fragments of varying sizes. The PrimeScript RT-PCR Kit is a 2-step RT-PCR kit featuring PrimeScript RTase, a reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through any RNA template containing higher-order structures. This enzyme works well on challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas up to 12 kb in length with good yield. The kit also includes TaKaRa s Ex Taq HS for efficient, sensitive, and specific amplification. The combination of PrimeScript with TaKaRa Ex Taq HS results in reliable, efficient, reproducible RT-PCR amplification from a large variety of RNA templates.regions or complex structures. This kit includes all reagents necessary for the reverse transcription of RNA to cdna and cdna amplification for end-point PCR. Kit Components (50 reactions): #RR014A PrimeScript RTase (for 2-step) 5X PrimeScript Buffer RNase Inhibitor 40 U/μL dntp Mixture 10 mm each Oligo dt Primer 2.5 μm Random 6 mers 20 μm TaKaRa Ex Taq HS 5 U/μL 10X PCR Buffer II Control F-1 Primer** 20 μm Control R-1 Primer*** 20 μm Positive Control RNA copies/μl RNase Free dh 2 O * 50 reactions of [Reverse transcription 20 μl g PCR 50 μl] ** Upstream sense primer for Positive Control RNA *** Downstream anti-sense primer for Positive Control RNA Not available in the United States 25 μl 200 μl 25 μl 150 μl 50 μl 50 μl 25 μl 250 μl 10 μl 10 μl 20 μl 1 ml PCR PRODUCTS 1 RT-PCR PrimeScript One Step RT-PCR Kit, Version 2 PrimeScript RTase Cat.# RR055A 50 reactions PrimeScript RTase Cat.# RR014B 200 reactions Powerful One Step RT-PCRs Features Robust and Efficient: PrimeScript 1 step Enzyme Mix with TaKaRa Ex Taq HS featuring specific and sensitive amplification The PrimeScript One Step RT-PCR Kit, Ver. 2 is a single-tube RT-PCR kit featuring PrimeScript RTase, a reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through higher-order RNA template structures. This enzyme works well on challenging templates at 50 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas of up to 12 kb in length with good yield. The kit also includes TaKaRa s Ex Taq HS for efficient, sensitive, and specific amplification. The combination of PrimeScript with TaKaRa Ex Taq HS results in reliable, efficient, reproducible RT-PCR amplification from a large variety of RNA templates. The single-tube, single step format utilizing the premix components (PrimeScript One Step Enzyme Mix, 2X One Step Buffer) provides simple and efficient reaction assembly. It also eliminates the need for reagent addition during the reaction process, minimizing the risk of contamination. Kit Components (50 reactions): #RR055A PrimeScript One Step Enzyme Mix 2X One Step Buffer Control F-1 Primer* 20 μm Control R-1 Primer** 20 μm Positive Control RNA 2 x 105 copies/μl RNase Free dh2o * Upstream sense primer for Positive Control RNA ** Downstream anti-sense primer for Positive Control RNA Not available in the United States 100 μl μl 20 μl 20 μl 20 μl μl 23

25 1PCR PRODUCTS PrimeScript 1st Strand cdna Synthesis Kit Features Excellent Elongation: able to synthesize long cdnas (up to 12 kb) with good yield. Robust and Efficient: carries out reverse transcription at standard RT temperature (42 C), even when RNA template contains high-order structures. The PrimeScript 1st Strand cdna Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cdna from total or poly(a)+ RNA using PrimeScript RTase. PrimeScript RTase is a MMLV-based RTase which possesses excellent elongation ability and is capable of synthesis of cdna of up to 12 kb in length with good yield. In addition, this enzyme works well on even challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. First strand cdnas synthesized with this kit can be used for a variety of applications including second strand synthesis, hybridization, PCR amplification, and real-time PCR. Kit Components (50 reactions): #6110A PrimeScript RTase 200 U/μL 5X PrimeScript Buffer RNase Inhibitor 40 U/μL dntp Mixture 10 mm each Oligo dt Primer 50 μm Random 6 mers 50 μm RNase free dh 2 O Not available in the United States RNA PCR Kit, Version 3.0 Cat.# RR019A 100 reactions RNA PCR Kit, Version 3.0 Cat.# RR019B 200 reactions (A 2) Synthesis of Full Length 1st Strand cdna From Total or poly(a)+ RNA 50 μl 200 μl 25 μl 50 μl 50 μl 100 μl 1 ml RT-PCR PrimeScript II 1st Strand cdna Synthesis Kit PrimeScript II 1st strand cdna Synthesis Kit contains all the reagents necessary to synthesize 1st strand cdna from total or poly(a)+ RNA using PrimeScript II RTase. A major factor that interferes with cdna synthesis is non-specific binding of reverse transcriptase to higher structure RNA (RNA with complex structure or longer fragments as templates). In addition, non-specific elongation due to mis-priming of the RT can cause problems in RT-PCR or the production of full-length cdna. PrimeScript II RTase, which contains an accessory protein similar to PrimeScript RTase, is a reverse transcriptase which decreases these problems in cdna synthesis reactions. This kit is useful to produce high-quality, full-length cdna. Kit Components (50 reactions): #6210A PrimeScript II RTase 200 U/μL 5X PrimeScript II Buffer RNase Inhibitor 40 U/μL dntp Mixture 10 mm each Oligo dt Primer 50 μm Random 6mers 50 μm RNase free dh 2 O Not available in the United States PrimeScript II 1st Strand cdna Synthesis Kit Cat.# 6210A 50 reactions PrimeScript II 1st Strand cdna Synthesis Kit Cat.# 6210B 200 reactions (A 2) Synthesis of Full Length 1st Strand cdna From Total or poly(a)+ RNA 50 μl 200 μl 25 μl 50 μl 50 μl 100 μl 1 ml 24

26 RNA LA PCR Kit, Version 1.1 RNA LA PCR Kit, Version 1.1 Cat.# RR012A 100 reactions RT-PCR using AMV Reverse Transcriptase Amplification of Longer cdna Templates using TaKaRa LA Taq The RNA LA PCR Kit is designed to generate longer and more accurate RT-PCR products. The kit uses AMV RT XL as the reverse transcriptase, which allows for efficient synthesis of first-strand cdna up to 12 kb. AMV RT XL also provides better thermostability than other reverse transcriptases, and retains its activity over a wider range of reaction temperatures. In addition, the kit uses LA Taq, which can synthesize products up to 40 kb in size with 6.5X better fidelity than Taq Polymerase. Both the reverse transcription and amplification reactions can be performed in a single tube. The components for 3 -RACE are also supplied. RNA PCR Kit, Version 3.0 RT-PCR for Analysis of Gene Expression at the RNA Level in a Conventional Two-Step, Single-Tube Configuration The RNA PCR Kit (AMV) Version 3.0 is designed for performing single tube reverse transcription of RNA to cdna using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase, with subsequent cdna amplification using TaKaRa Ex Taq HS. Taking advantage of AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42 60 C) than M-MLV RTase, this kit allows transcription at higher temperatures, which eliminates potential RNA secondary structure that can give rise to polymerase pauses. The supplied Oligo dt-adaptor Primer is designed for highly efficient cdna synthesis from the poly(a)+ RNA 3 -terminus, which allows amplification of unknown 3 -termini using 3 -RACE. The components for the 3 -RACE method are supplied in the kit. Kit Components RR012A AMV Reverse Transcriptase XL* (5 U/µL) 50 µl RNase Inhibitor (40 U/µL) 60 µl Random 9-mers (50 pmol/µl) 50 µl Oligo(dT) 20 -M4 Adaptor Primer (2.5 pmol/µl) 50 µl RNase-Free dh 2 O (DEPC-treated) 1 ml TaKaRa LA Taq (5 U/µL) 25 µl M13 Primer M4 (20 pmol/µl) 50 µl 10X RNA PCR Buffer 120 µl 10X LA Buffer II (without Mg 2+ ) 500 µl dntp Mixture (10 mm each) 150 µl MgCl 2 (25 mm) 1 ml Control Primer R-1 (20 pmol/µl) 25 µl Control Primer R-2 (20 pmol/µl) 25 µl Positive Control RNA ( copies/µl) 25 µl * Manufactured by Life Science Inc. 20 C Related Products AMV Reverse Transcriptase XL for RT-PCR (Cat.# 2630A) Ribonuclease Inhibitor (Cat.# 2313A) RNA PCR Kit, Version 3.0 Cat.# RR019A 100 reactions RNA PCR Kit, Version 3.0 Cat.# RR019B 200 reactions (A 2) Kit Components RR019A AMV Reverse Transcriptase XL* (5 U/µL) 50 µl RNase Inhibitor (40 U/µL) 60 µl Random 9-mers (50 pmol/µl) 50 µl Oligo (dt) Adaptor primer (2.5 pmol/µl) 50 µl RNase-Free dh 2 O 1 ml TaKaRa Ex Taq HS (5 Units/ µl) 25 µl M13 primer M4 (20 pmol/µl) 50 µl 10X RT Buffer (100 mm Tris-HCl, ph 8.3, 500 mm KCl) 1 ml 5X PCR Buffer 1 ml dntp Mixture (ea. 10 mm) 150 µl MgCl 2 (25 mm) 1 ml Control R-1 primer (20 pmol/µl) 25 µl (downstream primer for positive control RNA) Control F-1 primer (20 pmol/µl) 25 µl (upstream primer for positive control RNA) Positive control RNA ( copies/µl) 25 µl (transcribed poly(a) + RNA of psptet3 plasmid) * Manufactured by Life Science Inc. Positive control RNA: SP6 RNA polymerase was used to transcribe the supplied control RNA in vitro from plasmid psptet3, which contained a cloned 1.4 kb tetracycline resistance gene from pbr322. The control RNA is a poly(a+) RNA with a 30 base poly(a) tail. Plasmids cloned with a full-length double-stranded cdna prepared from this control RNA confer tetracycline resistance. Primer Primer Sequences: Random 9-mers: 5 -NNNNNNNNN-3 Oligo dt-adaptor Primer: The original primer includes dt and complementary region to M13 primer M4. Control F-1 primer: 5 -CTGCTCGCTTCGCTACTTGGA-3 Control R-1 primer: 5 -CGGCACCTGTCCTACGAGTTG-3 M13 primer M4: 5 -GTTTTCCCAGTCACGAC-3 Primers and Positive Control RNA are the same ones supplied in the RNA LA PCR Kit 20 C PCR PRODUCTS 1 RT-PCR 25

27 1PCR PRODUCTS RT-PCR One Step RNA PCR Kit One Step RNA PCR Kit Cat.# RR024A 50 reactions One Step RNA PCR Kit Cat.# RR024B 100 reactions (A 2) Reverse Transcription (RT) PCR from RNA Templates in a Single-Tube, One Step Reaction Takara s One Step RNA PCR Kit offers the convenience and time savings of performing reverse transcription and PCR in a single tube, without the need for changing buffers or adding reagents, minimizing the risk of contamination. AMV Reverse Transcriptase XL provides greater thermostability than other reverse transcriptases, and retains its activity over a wider range of reaction temperatures. The kit also contains AMV-Optimized Taq DNA Polymerase for efficient amplification of the cdna synthesized by AMV Reverse Transcriptase XL. M RT-PCR Amplification from Total RNA using the One Step RNA PCR Kit. RNA was isolated kb from HL60 cells and used as a template for RT-PCR with the One - 2 kb Step RNA PCR Kit. The primers - 1 kb chosen amplified products of 1 kb (lane 1), 2 kb (lane 2), and 4.4 kb (lane 3). Lane M contains l-hind III DNA Markers. Kit Components RR024A AMV Reverse Transcriptase XL* (5 U/µL) 50 µl AMV-Optimized Taq DNA Polymerase (5 U/µL) 50 µl RNase Inhibitor (40 U/µL) 60 µl RNase-Free dh 2 O (DEPC-treated) 2 1 ml 10X One-Step RNA PCR Buffer 250 µl dntp Mixture (10 mm each) 250 µl MgCl 2 (25 mm) 500 µl Control F-1 Primer (20 pmol/µl) 25 µl Control R-1 Primer (20 pmol/µl) 25 µl Positive control RNA ( copies/µl) 25 µl * Manufactured by Life Science Co. 20 C Related Products dntp Mixture (Cat.# 4030) Ribonuclease Inhibitor (Cat.# 2313) mrna Selective PCR Kit Version 1.1 mrna Selective PCR Kit Version 1.1 Cat.# RR025A 50 reactions Selective Amplification of cdna in the Presence of Genomic DNA This kit is designed to amplify only cdna that has been reverse-transcribed from RNA. This is achieved by selecting cdna in which a dntp analog has been incorporated during cdna synthesis. The kit enables successful cdna synthesis from RNA by using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase XL and cdna amplification by using AMV-Optimized Taq developed from LA (Long and Accurate) technology. The reactions are performed in a single tube (one step). This kit includes all the reagents required for cdna synthesis from RNA and cdna amplification. 20 C Related Products TaKaRa Ex Taq HS (Cat.# RR006) AMV Reverse Transcriptase XL for RT-PCR (Cat.# 2630A) Ribonuclease Inhibitor (Cat.# 2313) Kit Components RR025A AMV Reverse Transcriptase XL (5 U/µL)* 50 µl RNase inhibitor (40 units/µl) 50 µl AMV-Optimized Taq (5 units/µl) 50 µl 2x mrna Selective PCR Buffer I** ml 2x mrna Selective PCR Buffer II** ml MgCl 2 (25 mm) 500 µl dntp/analog mixture*** 250 µl Control F-1 primer (20 µm) 10 µl Control R-1 primer (20 µm) 10 µl Control Template 10 µl RNase-Free dh 2 O 1 ml Random 9-mers (50 µm) 50 µl Oligo dt Primer (50 µm) 50 µl * Manufactured by Life Sciences, Inc. ** We generally recommend the use of Buffer I. However, in cases of low yield or no amplification products due to low GC content in the target DNA, Buffer II is recommended. *** dntp analog mixture is prepared by adding dntp analog to a mixture of four dntps (each dntp at 10 ml concentration). 26

28 RACE Core Set, 3'-Full and 5'-Full RACE Core Set, 3 -Full Cat.# reactions RACE Core Set, 5 -Full Cat.# reactions Rapid Amplification of cdna Ends, Cloning Full-Length cdna Sequences Reverse transcription of mrna followed by PCR is a common method for obtaining cdna clones for expression analysis. However, it can be challenging to obtain full-length cdna by this method. Rapid amplification of cdna ends (RACE) helps overcome this problem by using a gene-specific primer for reverse transcription, followed by amplification with specially designed oligonucleotide primers. The 3 -Full and 5 -Full RACE Core Sets uses inverse PCR to amplify an unknown 3 - or 5 -end of a cdna. The kit contains all the reagents needed for reverse transcription, degradation of the DNA-RNA hybrid, circularization of single-stranded cdna and subsequent amplification. The kit uses AMV Reverse Transcriptase XL to maximize the length of the first-strand cdna, and is recommended for use with TaKaRa s Taq, Ex Taq, or LA Taq. BcaBEST RNA PCR Kit BcaBEST RNA PCR Kit Cat.# RR023A 100 reactions BcaBEST RNA PCR Kit Cat.# RR023B 200 reactions (A 2) Single-tube RT-PCR, using LA PCR Technology for Better PCR Performance Reverse Transcription of RNAs with High Amounts of Secondary Structure The Bca BEST RNA PCR Kit is designed to perform both reverse transcription and DNA amplification in a single tube. The kit uses Bca BEST DNA Polymerase, which contains both DNA polymerase and reverse transcriptase for first strand cdna synthesis. In contrast to standard reverse transcriptases, BcaBEST Polymerase has a temperature optimum of 65 C, which permits cdna synthesis from difficult and highly structured RNA templates. Bca-Optimized Taq Polymerase is used for second strand synthesis and subsequent PCR. This polymerase utilizes LA PCR technology for improved PCR length and accuracy. Random 9-mers, oligo-dt primers, or a specific downstream primer that acts as an antisense primer in PCR can be used for cdna synthesis. Kit Components 6122 AMV Reverse Transcriptase XL* (5 U/µL) 10 µl RNase Inhibitor (40 U/µL) 10 µl 10X RT Buffer (containing dntp mix) 15 µl RNase H (60 U/µL) 10 µl 5X Hybrid RNA Degradation Buffer 150 µl T4 RNA Ligase (40 U/µL) 10 µl 5X RNA (ssdna) Ligation Buffer 80 µl 40% PEG µl RNase-Free dh 2 O (DEPC-treated) 1 ml Positive Control RT Primer (200 pmol/µl) 10 µl Positive Control 1st Primer Pair (ea. 20 pmol/µl) 10 µl Positive Control 2nd Primer Pair (ea. 20 pmol/µl) 10 µl Positive Control RNA (10 ng/µl) 10 µl *Manufactured by Life Science Inc AMV Reverse Transcriptase XL* (5 U/µL) 20 µl RNase Inhibitor (40 U/µL) 10 µl 10X RNA PCR Buffer 40 µl dntp Mixture (10 mm each) 40 µl MgCl 2 (25 mm) 80 µl RNase-Free dh 2 O (DEPC-treated) 500 µl Oligo dt-3 Sites Adaptor-Primer (2.5 µm) 20 µl 3 Sites Adaptor Primer (20 µm) 20 µl Control F-1 3 Sites Adaptor Primer (20 µm) 10 µl Positive Control RNA ( copies/µl) 10 µl 20 C Related Products TaKaRa PCR Polymerases dntp s (Cat.# ) Kit Components RR023A BcaBEST DNA Polymerase (22 U/µL) 50 µl RNase Inhibitor (40 U/µL) 25 µl Random 9-mers (50 µm) 50 µl Oligo dt Primer (50 µm) 50 µl RNase-Free dh 2 O (DEPC-treated) 1 ml Bca-Optimized Taq (5 U/µL) 25 µl 2X Bca 1st Strand Buffer ml 5X Bca 2nd Strand Buffer 800 µl dntp Mixture (10 mm each) 50 µl MgSO 4 (25 mm) 500 µl Control F-1 Primer (20 µmol/µl) 25 µl Control R-1 Primer (20 µmol/µl) 25 µl Positive Control RNA (1 µg/µl) 25 µl 20 C Related Products dntp Mixture (Cat.# 4030) AMV RT (Cat.# 2630A) Ribonuclease Inhibitor (Cat.# 2313) PCR PRODUCTS 1 General PCR Products 27

29 Introduction to Real Time PCR (qpcr) 1PCR PRODUCTS Real time PCR is a method for quantitative fluorescent detection of the initial amounts of DNA template in a sample. Real time PCR (also called qpcr, for quantitative PCR) requires the use of a specialized thermocycler designed to detect the emissions of amplified fluorescently-labeled DNA molecules. This technique has been used for many diverse applications, including the detection of pathogenic bacteria, identification and quantitation of microorganisms from water samples, and detection of SNPs (single nucleotide polymorphisms) in genomic sequences, just to name a few. Two common fluorescent-based DNA detection methods used for Real Time PCR include: 1) direct labeling of dsdna by SYBR Green I, or 2) probe detection method using the 5 nuclease assay commonly referred to as the TaqMan Probe Method (see figures below). Real Time PCR Probe Detection Method SYBR Green I Detection Method Guide to qpcr Polymerases Real Time PCR Polymerase Premix Ex Taq (Probe qpcr) * SYBR Premix Ex Taq (Tli RNase H+)* SYBR Premix Ex Taq II (Tli RNase H+)* SYBR Premix DimerEraser (Perfect Real Time)* Amplification Efficiency Fidelity Proofreading Activity Specificity Convenience GC-Rich Templates Hot-Start PCR Guidelines for Length of Primers Terminal Transferase Activity (3 -A overhang) Taq** Yes bp Yes Taq** Yes bp Yes Taq** Yes bp Yes Taq** Yes bp Yes * Sample Available ** All fidelity determined by using the Kunkel method. 28

30 Comparison of TaKaRa s Real Time PCR and qrt-pcr Kits SYBR Premix Ex Taq (Tli RNase H+) Premix Ex Taq (Probe qpcr) SYBR Premix Ex Taq II (Tli RNase H+) Cat.# RR420A RR390A RR820A Reactions/Kit 200 (50 µl reaction size) 200 (50 µl reaction size) 200 (50 µl reaction size) Premix or Separates Premix Premix Premix Reaction Volumes Smart Cycler ABI LightCycler 25 µl 10, 25, 50 µl 20 µl 25 µl 20, 50 µl 20 µl Not Applicable 10, 25, 50 µl 20 µl Enzyme TaKaRa Ex Taq Hot Start TaKaRa Ex Taq Hot Start TaKaRa Ex Taq Hot Start Buffer 2X premix with optimized buffer (with Mg 2+ ) (Tli RNaseH) (note: buffer differs from RR390) 2X premix with optimized buffer (with Mg 2+ ) (note: buffer differs from RR420) 2X premix with optimized buffer (with Mg 2+ ) (note: buffer differs from RR420) Detection Method SYBR Green I (supplied in premix) SYBR Green I or TaqMan Probes (not supplied) SYBR Green I (supplied in premix) Reference Dye ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) Instruments Supported* Applied Biosystems 7300/7700/7900 HT/7500 real time PCR system, StepOne Plus real time PCR system (Life Technologies) icycler, LightCycler, Smart Cycler, Mx3000P Note: All three enzymes include Tli Rnase H, a heat resistant RNase H Applied Biosystems 7300/7700/7900 HT/7500 real time PCR system, StepOne Plus real time PCR system (Life Technologies) LightCycler, Smart Cycler, Smart Cycler II, icycler Applied Biosystems 7300/7500 real time PCR system, StepOne Plus real time PCR system (Life Technologies) LightCycler, Smart Cycler, Smart Cycler II One Step PrimeScript Reagent Kit One Step PrimeScript RT-PCR Kit (Perfect Real One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) Time) (Perfect Real Time) Cat.# RR037A RR064A RR066A Reactions/Kit 200 (10 µl reverse transcription) 100 (50 µl reaction) 100 (50 µl reaction) Premix or Separates Buffer + Enzyme Mix Separate components Separate components Reaction Volumes Smart Cycler ABI LightCycler Enzyme Buffer Detection Method 10 µl 20, 50 µl 10 µl PrimeScript RTase + RNase Inhibitor mixed (mix II) 5X PrimeScript Buffer (for Real Time) including Mg 2+ and dntp Mixture SYBR Green I or TaqMan Probes using RR041A or RR039A respectively 25 µl 20, 50 µl 20 µl PrimeScript Enzyme mix II +TaKaRa Ex Taq HS 2X One Step RT-PCR Buffer III, including Mg 2+ and dntp Mixture SYBR Green I or TaqMan Probes (not supplied) 25 µl 20, 50 µl 20 µl PrimeScript Enzyme mix II +TaKaRa Ex Taq HS 2X One Step SYBR RT-PCR Buffer III, including Mg 2+ and dntp Mixture SYBR Green I Reference Dye N/A ROX & ROX II (as separate tubes) ROX & ROX II (as separate tubes) Instruments Supported* ABI PRISM 7000/7700 Applied Biosystems 7300/7500 icycler LightCycler MJ Opticon MX3000 RotorGene Smart Cycler Thermal Cycler Dice Real Time System Thermal Cycler Dice Real Time System ABI PRISM 7000/7700/7900 HT Applied Biosystems 7300/7500 LightCycler Smart Cycler Smart Cycler II Thermal Cycler Dice Real Time System ABI PRISM 7000/7700/7900 HT Applied Biosystems 7300/7500 LightCycler Smart Cycler Smart Cycler II PCR PRODUCTS 1 Real Time PCR EASY Dilution Solution (for real time PCR) EASY Dilution Solution (for real time PCR) Cat.# ml Dilute Template DNA or RNA used in Standard for Standard Curve This product is used to dilute template DNA or RNA when preparing serial dilutions of the standard for establishing the standard curve in real time PCR. 20 C. Store this product in aliquots to avoid contamination. 29

31 1PCR PRODUCTS Real Time PCR SYBR Premix Ex Taq (Tli RNase H Plus) SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420A 200 reactions SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420B 400 reactions (A size 2) SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420L 200 reactions SYBR Premix Ex Taq (Tli RNase H Plus) Cat.# RR420W 1000 reactions (L size 5) Features RNase H included in Reaction Mix: Reduces inhibition from mrna/cdna hybrids Eliminates need for RNase H Digestion Step: When using low RNase H RTs Amplification of Longer Fragments: Able to amplify up to 570 bp fragments High Sensitivity: Detects as few as 100 copies Accurate Quantitation: Excellent standard curves for various real time instruments have been established Instrument Compatibility: Compatible with all qpcr instruments SYBR Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double stranded RNA/cDNA hybrids remaining after cdna synthesis. This inhibition is common when using low or RNase H minus RTs, and with GC rich templates and/or genes with poor expression. The presence of dsrna/cdna hybrids is a frequent cause of poor qpcr amplification and/or reaction failure. SYBR Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qpcr. Long Target qpcr Reactions Kit Components RR420A SYBR Premix Ex Taq (Tli RNase H Plus) (2X conc.) *1 5 1 ml ROX Reference Dye (50X conc.) *2 200 µl ROX Reference Dye II (50X conc.) *2 200 µl RR420L SYBR Premix Ex Taq (Tli RNase H Plus) (2X conc.) *1 1 5 ml ROX Reference Dye (50X conc.) *2 200 µl ROX Reference Dye II (50X conc.) *2 200 µl *1 Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, Tli RNaseH and SYBR Green I. *2 This component is to be used for analyses using a device that corrects fluorescent signals between wells, such as the real-time PCR device by Applied Biosystems. Please use ROX Reference Dye for Applied Biosystems 7900HT/7300 Real-Time PCR System or StepOnePlus and ROX Reference Dye II for 7500 Real-Time PCR System or 7500 Fast Real-Time PCR System. ROX Reference Dye II is compatible with Agilent Mx3000P. This component is not required with Thermal Cycler Dice Real Time System II, Smart Cycler System or LightCycler. 4 C Note : Product is stable for up to 6 months when stored at 4 C. Protect this kit from light and avoid contamination. This kit is shipped in a package with dry ice. Store the kit at 4 C and in darkness. For long-term storage, store at 80 C. (Do not store at 20 C) Store a thawed product at 4 C and use within 6 months. Cq=12 SYBR Premix Ex Taq (Tli RNase H+)(Cat.# RR420) Cq=22 Company A SYBR Master Mix Target: ACTB (533 bp). Results: Superior performance on long fragments and excellent Cq values when using SYBR Premix Ex Taq (Tli RNase H+)(Cat.# RR420) Greater Results with Longer Amplicons: amplify targets up to ~533 bp 30

32 SYBR Premix Ex Taq II (Tli RNaseH Plus) SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820A 200 reactions SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820B 400 reactions (A size 2) SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820L 200 reactions SYBR Premix Ex Taq II (Tli RNase H Plus) Cat.# RR820W 400 reactions (A size 2) Features Optimized for Real Time PCR with SYBR Green I Easy Preparation Extremely High Specificity Minimization of PCR Inhibition: due to residual mrna by premixed Tli RNase H SYBR Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using SYBR Green I. It is supplied at a 2X concentration premixed with SYBR Green I at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mrna when using cdna as template. This product, with a modified buffer composition, offers a higher reaction specificity than that by SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and TaKaRa Ex Taq HS, a hot start PCR enzyme that uses an anti-taq antibody, allows highly reproducible and reliable real time PCR analyses. Compatible instruments include : Applied Biosystems 7300/7500 Real-Time PCR System, 7500 Fast Real-Time PCR System, StepOnePlus Real-Time PCR System (Life Technologies) LightCycler (Roche Diagnostics) Note: We recommend using SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A) for Smart Cycler System/Smart Cycler II System (Cepheid). TAKARA BIO is under a license agreement with Molecular Probes Inc. for the use of SYBR Green I as a reagent for research purposes. SYBR is a registered trademark of Molecular Probes Inc. Kit Components RR820A SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X conc.) *1 5 1 ml ROX Reference Dye (50X conc.) *2 200 µl ROX Reference Dye II (50X conc.) *2 200 µl RR820L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X conc.) *1 1 5 ml ROX Reference Dye (50X conc.) *2 200 µl ROX Reference Dye II (50X conc.) *2 200 µl *1 Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, Tli RNaseH and SYBR Green I. *2 This component is to be used for analyses using a device that corrects fluorescent signals between wells such as the real-time PCR device by Applied Biosystems. Please use ROX Reference Dye for Applied Biosystems 7300 Real-Time PCR System or StepOnePlus and ROX Reference Dye II for 7500/7500 Fast Real-Time PCR System. This component is not required with Thermal Cycler Dice Real Time System II or LightCycler. 4 C Note : Product is stable for up to 6 months when stored at 4 C. Protect this kit from light and avoid contamination. This kit is shipped in a package with dry ice. Store the kit at 4 C and in darkness. For long-term storage, store at 80 C. (Do not store at 20 C) Store a thawed product at 4 C and use within 6 months. PCR PRODUCTS 1 Real Time PCR Best Performance on Standard Targets Target: ACTB (186 bp) Results: Superior Specificity, maintains signal below baseline in no template control reactions. SYBR Premix Ex Taq II (Tli RNase H+)(Cat.# RR820) Company A SYBR Master Mix Superior Specificity: limits primer dimers and off-target amplification 31

33 1PCR PRODUCTS Real Time PCR Premix Ex Taq (Probe qpcr) Premix Ex Taq (Probe qpcr) Cat.# RR390A 200 reactions ( 50 µl PCR) Premix Ex Taq (Probe qpcr) Cat.# RR390B 400 reactions ( 50 µl PCR) (A size x 2) Premix Ex Taq (Probe qpcr) Cat.# RR390L 200 reactions ( 50 µl PCR) Premix Ex Taq (Probe qpcr) Cat.# RR390W 1000 reactions ( 50 µl PCR) (L size x 5) Real Time PCR (qpcr) Quantitation of DNA using Probe Detection Features Rapid and Accurate Detection and Quantitative Gene Expression Analysis: using real-time PCR 2X Premix: makes pipetting easy Excellent Amplification Efficiency and Highly Sensitive Detection: from the optimized buffer Includes Tli RNaseH, a Heat-Resistant RNaseH: to minimize PCR inhibition by residual mrna in reactions using a cdna template Premix Ex Taq (Probe qpcr) is a 2X premix for real-time PCR (qpcr) detection with TaqMan. The 2X Premix TaKaRa Ex Taq HS, a hot start PCR enzyme with an anti-taq antibody, and a buffer optimized for real-time PCR. The resulting premix allows excellent suppression of non-specific amplification, high amplification efficiency, and high detection sensitivity in real-time PCR analyses. In addition, Tli RNaseH, a heatresistant RNaseH, is included to minimize PCR inhibition from residual mrna in reactions using cdna templates. This product is excellent for high-speed PCR and allows accurate target quantification and detection over a broad dynamic range, making it possible to conduct highly reproducible and reliable real-time PCR analyses. Store at 4 C (stable for up to 6 months). *Every precaution should be taken to avoid contamination. This product is shipped frozen at 20 C. Store the product at 4 C after receipt. Before use, gently invert tube to make sure reagent is completely dissolved and evenly mixed. This product may be frozen at 20 C for long term storage. Once thawed, it should be stored at 4 C and used within 6 months. Kit Components RR390A Premix Ex Taq (Probe qpcr) (2 conc.) *1 5 1 ml ROX Reference Dye (50 conc.) *2 200 µl ROX Reference Dye II (50 conc.) *2 200 µl RR390L Premix Ex Taq (Probe qpcr) (2 conc.) *1 1 5 ml ROX Reference Dye (50 conc.) *2 200 µl ROX Reference Dye II (50 conc.) *2 200 µl *1 Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, and Tli RNase H. *2 Use when performing analyses with a device such as real-time PCR instruments by Life Technologies that normalize fluorescent signals between wells. Use ROX Reference Dye for ABI PRISM 7000/7700, 7300 Real-Time PCR System, and StepOnePlus Real-Time PCR System. Use ROX Reference Dye II for 7500 Real-Time PCR System and 7500 Fast Real-Time PCR System. Use the ROX Reference Dye at a final concentration of 1X and the ROX Reference Dye II at a final concentration of 0.5X. No dye is required when using Thermal Cycler Dice Real Time System II, Smart Cycler System, or LightCycler. Best Performance with TaqMan Probe Data using the Premix Ex Taq (Probe qpcr) product on the Applied Biosystems StepOnePlus instrument and the Roche LightCycler 480 is shown. Template: mouse liver cdna (equivalent total RNA 1 pg ~ 100 ng) Target: Yeast (using TaqMan Gene Expression Assays) Applied Biosystems StepOnePlus Roche LightCycler 480 R 2 = PCR Efficiency= 101.0% R 2 = PCR Efficiency= 100.0% 32

34 SYBR Premix DimerEraser (Perfect Real Time) SYBR Premix DimerEraser (Perfect Real Time) Cat.# RR091A 200 reactions SYBR Premix DimerEraser (Perfect Real Time) Cat.# RR091B 400 reactions (A size x 2) For Real time PCR Quantification of DNA using SYBR Green I Features Quick and accurate detection and quantification of target gene through real time PCR Easy-to-use 2X premix reagent including SYBR Green I: Ready to perform real time PCR in the presence of a fluorescent intercalater. Just add PCR primers, template and dh2o to start the reaction. High amplification efficiency and highly sensitive detection: This product utilizes an enzyme for hot start, TaKaRa Ex Taq HS. As this enzyme buffer system is optimized for real time PCR, this product offers highly efficient amplification and highly sensitive detection. Moreover, the addition of the accessory protein strongly suppresses mis-priming of primers during PCR reaction and nonspecific amplification, such as primer-dimer. SYBR Premix DimerEraser (Perfect Real Time) is a real-time PCR specific reagent using the SYBR Green I 1 intercalater method. The reagent is supplied as a 2X premix making preparation of the reaction mixture easier. This product has an improved buffer system which results in improved reaction specificity. It effectively depresses primer dimers, which is especially important in SYBR Green Assay. SYBR Premix DimerEraser (Perfect Real Time) also makes it possible to measure accurate quantitative analysis over a broad range of template concentration by suppression of non-specific amplification and more sensitive detection. We recommend a 3-step PCR method in the standard protocol of this product. Applicable Real Time PCR Instruments ABI PRISM , Applied Biosystems 7500 Real-Time PCR System, 7500 Fast Real- Time PCR System (Applied Biosystems) LightCycler (Roche Diagnostics) 3 Thermal Cycler Dice Real Time System (TAKARA BIO) (Note: This instrument is not available in the U.S. or Europe.) Smart Cycler System/Smart Cycler II System (Cepheid) 4 1. SYBR Green I is provided under licensing agreement with Molecular Probes Inc. for internal research use only. SYBR is a registered trademark of Molecular Probes Inc. 2. ABI PRISM is a registered trademark of Applera Corporation. 3. LightCycler is a registered trademark of Roche Diagnostics. 4. Smart Cycler is a registered trademark of Roche Cepheid. Kit Components RR091A SYBR Premix DimerEraser (Perfect Real Time) (2X conc.) ml ROX Reference Dye (50X conc.) μl ROX Reference Dye II (50X conc.) μl 1. Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, and SYBR Green I. Refer to IX. Specifications for further details of TaKaRa Ex Taq HS. 2. ROX Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM 7000, the use of ROX Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System and 7500 Fast Real-Time PCR System, the use of ROX Reference Dye II is recommended. It is not required for use with LightCycler, Smart Cycler System/Smart Cycler II System and Thermal Cycler Dice Real Time System. Reagents and Instruments Required but Not Supplied with this Product Thermal Cycler for real time PCR Reaction tube or plate for real time PCR PCR primer dh2o Micropippets and Micropippet tips (autoclaved prior to use) Stable at 4 C for 6 months. (Note: product should be protected from light. Avoid contamination.) This product is shipped on dry ice and on delivery should be stored at 4 C and protected from light. Make the concentration uniform by gently inverting a tube before use. For longer-term storage, 80 C is recommended. Notes TaKaRa Ex Taq HS used in this product is an enzyme for hot start PCR utilizing a Taq antibody. The initial denaturation step prior to PCR should be 95 C for 30 sec. It does not require the activation step of heating at 95 C for min prior to the PCR. SYBR Premix DimerEraser (Perfect Real Time) does not contain Tli RNase H. Transgene Detection Primer Set for Real Time (Mouse) Transgene Detection Primer Set for Real Time (Mouse) Cat.# reactions PCR PRODUCTS 1 Real Time PCR Detection and Quantification of Transgene Inserted in Transgenic Mouse Genome This kit is a primer set for real-time PCR to detect GFP (EGFP, AcGFP1) and lacz, which are gene markers in transgenic mice. It is possible to screen transgenic mice by detecting these marker genes with real-time PCR. The primer sets (Ywhaz, Raver2) for detecting genes on mouse genomic DNA are also included as a reference. Therefore, it is possible to make a comparison of the transgene contents between samples by relative quantification. Kit Components 3788 GFP Primer-1 (2 μm ea.) μl GFP Primer-2 (2 μm ea.) μl lacz Primer-1 (2 μm ea.) μl lacz Primer-2 (2 μm ea.) μl Reference Primer-1 (2 μm ea.) μl Reference Primer-2 (2 μm ea.) μl 1 GFP Primer-1 is designed for consensus sequence common to both EGFP and AcGFP1 2 GFP Primer-2 is designed for EGFP. It is not used for AcGFP1 detection 3 lacz primer -1 and -2 are designed for lacz (β-galactosidase) gene and positioned at different sites 4 Reference Primer-1 is for a region of Ywhaz gene on mouse chromosome 15 5 Reference Primer-2 is for a region of Raver2 gene on mouse chromosome C 33

35 1PCR PRODUCTS One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) Not available in the United States One Step SYBR PrimeScript RT-PCR Kit Cat.# RR066A 100 reactions One Step SYBR PrimeScript RT-PCR Kit Cat.# RR066B 500 reactions (A size x 5) Features Easy & Efficient: One Step RT-PCR lowers pipeting and contamination risks. Sensitive: Accurate quantification of any RNA. One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) is designed for real time, one-step RT-PCR using SYBR Green I (1)(2) detection and is suitable for detection of small amounts of RNA, i.e., viral RNA. In this kit, RT-PCR can be performed in a single tube minimizing contamination. Also, amplified products are monitored in real time, so there is no need to verify results through electrophoresis after PCR. This kit uses PrimeScript RTase, a robust reverse transcriptase which can quickly and efficiently synthesize cdna, and TaKaRa s Ex Taq HS, a high efficiency hot start PCR enzyme. These enzymes have been optimized for One Step RT-qPCR. Kit Components RR066A 2X One Step SYBR RT-PCR Buffer III* TaKaRa Ex Taq HS 5U /μl PrimeScript RT Enzyme Mix II** RNase Free dh 2 O ROX Reference Dye 50X conc.*** ROX Reference Dye II 50X conc.*** * Includes dntp Mixture, Mg 2+ and SYBR Green I ** Includes RNase Inhibitor and RTase μl 100 μl 100 μl ml 100 μl 100 μl Note *** ROX Reference Dye/Dye II is used for normalization of fluorescence intensity by background subtraction. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 SYBR Green I is licensed by Molecular Probes Inc. for research reagents. SYBR is a registered trademark of Molecular Probes Inc.. 2 Use One Step PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using TaqMan probe for detection. TaqMan is a registered trademark of Roche Molecular Systems Inc. Real Time PCR One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) Not available in the United States One Step SYBR PrimeScript RT-PCR Kit II Cat.# RR086A 100 reactions One Step SYBR PrimeScript RT-PCR Kit II Cat.# RR086B 500 reactions (A size x 5) Features Easy & Efficient: One Step RT-PCR lowers pipeting and contamination risks. Specificity: Improved buffer and hot start polymerase. Sensitive: Accurate quantification of any RNA inclucing RNA viruses or small amounts of RNA One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) is designed for Real- Time One Step RT-PCR including SYBR Green I (1)(2). This kit is suitable for detection of minute amounts of RNA such as RNA viruses. This kit uses PrimeScript RTase, which has excellent elongation ability and can efficiently synthesize cdna in short periods of time, and TaKaRa Ex Taq HS high efficiency/hot start PCR enzyme. It is optimized for one step RT-PCR, combining the TaKaRa Bio RT-PCR technology with these enzymes allows an increase in the yield of RT-PCR product. This kit contains an improved buffer system which results in improved reaction specificity compared to the One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Cat.# RR066A). Premixed components allow simple and convenient reaction assembly. Kit Components RR086A TaKaRa Ex Taq HS 5 U/μL 2X One Step SYBR RT-PCR Buffer IV* 840 μl 3 PrimeScript 1 step enzyme Mix II** 200 μl RNase Free dh 2 O 1.25 ml 2 ROX Reference Dye*** 50X conc. 100 μl ROX Reference Dye II*** 50X conc. 100 μl * Includes dntp Mixture, Mg 2+ and SYBR Green I ** Includes PrimeScript RTase, RNase Inhibitor, TaKaRa Ex Taq HS Note *** ROX Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 SYBR Green I is licensed by Molecular Probes Inc. for research reagents. SYBR is a registered trademark of Molecular Probes Inc.. 2 Use One Step PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using TaqMan probe for detection. TaqMan is a registered trademark of Roche Molecular Systems Inc. 34

36 One Step PrimeScript RT-PCR Kit (Perfect Real Time) Not available in the United States One Step PrimeScript RT-PCR Kit Cat.# RR064A 100 reactions One Step PrimeScript RT-PCR Kit Cat.# RR064B 500 reactions (A size x 5) One Step RT-qPCR using the TaqMan Probes Features Robust and Efficient: RT-qPCR all in one tube with PrimeScript RT ensuring efficient transcription from any RNA One Step PrimeScript RT-PCR Kit (Perfect Real Time) is designed for real time one-step RT-qPCR using TaqMan probe(1,2) detection. In this kit, RT-PCR can be performed in a single tube, minimizing contamination and is suitable for detection of small amounts of RNA, such as a viral RNA. In addition, amplified products are monitored in real time, so there is no need to verify results through electrophoresis after PCR. Kit Components RR064A 2X One Step RT-PCR Buffer III * TaKaRa Ex Taq HS 5U/μL PrimeScript RT Enzyme Mix II ** RNase Free dh 2 O ROX Reference Dye *** 50X conc. ROX Reference Dye II *** 50X conc. * Includes dntp Mixture and Mg 2+. ** Includes RNase Inhibitor and RTase μl 100 μl 100 μl ml 100 μl 100 μl Note *** ROX Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX Reference Dye II is recommended. The use of ROX Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice Real Time System, Smart Cycler or LightCycler real time instruments. 1 TaqMan is a registered trademark of Roche Molecular Systems Inc. 2 Use One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) for real time one-step RT-PCR using SYBR Green I for detection. PCR PRODUCTS 1 PrimeScript RT Reagent Kit (Perfect Real Time) PrimeScript RT Reagent Kit (Perfect Real Time) Cat.# RR037A 200 reactions PrimeScript RT Reagent Kit (Perfect Real Time) Cat.# RR037B 800 reactions Application Robust Reverse Transcription Optimized for Real Time PCR Features Fast, Efficient cdna Synthesis for real time PCR possible. This kit is best suited for two-step, real-time RT-PCR. Includes Random 6 mers and Oligo dt Primer for use as reverse transcription primers. The reaction can be performed using a mixture of these two primers, or the primer can be selected based on the purpose of the experiment. Futhermore, gene specific primers can be used for specific gene detection. The PrimeScript RT-PCR Kit is a 2-step RT reagent kit featuring PrimeScript RTase, a new reverse transcriptase which offers robust reverse transcription on any RNA template. PrimeScript RTase is a MMLV-based RTase which provides complete elongation through any RNA template containing higher-order structures. It produces full-length cdnas of up to 12 kb in length with good yield. The kit is simple to use and is suitable for high through-put analysis. Kit Components RR037A 5X PrimeScript Buffer (for Real Time)* 400 µl PrimeScript RT Enzyme Mix I 100 µl Oligo dt Primer (50 µm) 100 µl Random 6 mers (100 µm) 100 µl RNase Free dh 2 O 1 ml EASY Dilution (for Real Time PCR)** 1 ml * Contains dntp Mixture and Mg 2+. ** This solution can be used to prepare the dilution series of total RNA or cdna for establishing a standard curve. In contrast to dilution with water or TE, EASY Dilution facilitates accurate low concentration dilution. The Easy Dilution does not inhibit either reverse transcription or PCR enzyme activity. The diluted template solutions can be used as the templates for reverse transcription or PCR reactions. EASY Dilution is also available separately (Cat.# 9160). Note Easy Dilution has been tested with TaKaRa Bio s real-time PCR reagent. Compatibility with products from other manufacturers has not yet been verified. 20 C Real Time PCR 35

37 1PCR PRODUCTS Real Time PCR CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) CellAmp Direct RNA Prep Kit RT-PCR (Real Time) Cat.# reactions Prepare Templates for Real-Time RT-PCR CellAmp Direct RNA Prep Kit RT-PCR is for preparation of templates used in onestep and two-step real-time RT-PCR utilizing a simple method of extraction directly from cultured cells in 96 well-plates or others. It is possible to prepare templates for real-time PCR from cultured cells in approximately 10 minutes. When using this kit with a One step real-time RT-PCR kit (i.e. One Step SYBR PrimeScript RT-PCR Kit), gene expression analysis can be performed in approximately 2 hours. Templates can be prepared directly from a small quantity of cells with this kit, facilitating gene expression analysis by sensitive real-time RT-PCR. Excellent for detection of rare transcripts. Kit Components 3732 CellAmp Washing Buffer 2 x 12.5 ml CellAmp Processing Buffer 10 ml DNase I for Direct RNA Prep 200 µl 20 C CellAmp Washing Buffer and CellAmp Processing Buffer can be stored at 4 C after thawing. Avoid contamination. Analytical Results on Expressions by 6 Different Genes. For all lysate samples, regardless of the number of cells from which they were prepared, both the 1-step and 2-step real-time RT-PCRs provided stable gene expression profiles similar to that obtained using the high-purity RNA sample prepared with the FastPure RNA Kit. Cell Amp Whole Transcriptome Amplification Kit (Real Time) Not available in the United States Cell Amp Whole Transcriptome Amplification Kit (Real Time) Cat.# assays Template preparation and amplification from few cells of pg RNA for qpcr analysis. Features Direct cdna amplification, from small amount of cells. Efficient amplification of cdna, without purifying RNA or cdna. Good representativity : little bias included by amplification. This kit is designed to perform cdna synthesis amplification directly from small amounts of cells. The amplified cdna can be applied for real-time PCR as a template. In general, there is a problem in loss of nucleic acids during the purification process when dealing with small amounts cells. Moreover, cdna can be amplified efficiently without purifying RNA or cdna by using this kit. First, in this kit, cell lysis is performed, followed by synthesiz of cdna from mrna by reverse transcription using dt adapter primer (RT dt Primer). Next, add da-tail by Terminal Deoxynucleotidyl Transferase (TdT) to synthesized cdna, which will be used as a template for low cycle PCR to amplify the cdna. This kit is useful to directly amplify cdna from cells, and also to amplify cdna from small amount of RNA. Takara s Ex Taq HS (RR006A) is recommanded for efficient and consistent amplification of low and highly represented cdna. dntp Mixture (2.5 mm each) 20 µl RT Enzyme Mix 1 50 µl TdT Buffer 300 µl datp (100 mm) 30 µl TdT Enzyme Mix 2 25 µl PCR Primer Mix 600 µl x 2 RNase Free dh 2 O 1 ml x 2 1 Contains PrimeScript Reverse Transcriptase and RNase Inhibitor 2 Contains Terminal Deoxynucleotidyl Transferase and RNase H 20 C Application example Amplification of cdna from mouse cell (2, 20, or 200 cells) and mouse total RNA (20 pg, 200 pg or 2 ng) were performed. The obtained cdna amplified products were diluted to 1/10, and 2 μ l of those was used as PCR template. The figures show the result of Mouse Rplp1 which was obtained by performing real-time PCR. The template used for real-time PCR corresponds to mouse cell (8 10-4, , cells) and mouse total RNA ( pg, pg, pg) when recalculated to initial template amount. This result shows that efficient amplification of cdna was performed. Mouse cell Kit Components 3730 Lysis Buffer 350 µl RNase Inhibitor (40 U/µl) 25 µl RT dt Primer 20 µl Total RNA from mouse liver Target: Mouse Rplp1 / Reagent: SYBR Premix Ex Taq II 36

38 CellAmp Direct RNA Prep Kit for Real Time PCR and Protein Analysis CellAmp Direct RNA Prep Kit for Real Time PCR & Protein Analysis Cat.# reactions Prepare Templates for Real-Time RT-PCR and Protein Analysis Features Economical, Easy to Use and Flexible Quick and Simple Preparation of RNA (10 min) Template: from cultured cells RNA can be used for One-Step or Two-Step qrt-pcr Works with SYBR or TaqMan Probe Detection Gene Analysis Performed in 2 hours: including RNA extraction and One Step qrt PCR reaction Can Use in Combination with Western Blot Analysis: loading buffer supplied for SDS-Page The CellAmp Direct RNA Prep Kit for qrt-pcr and Protein Analysis allows simple, direct extraction directly from cultured cells to prepare templates used for onestep and two-step qrt-pcr. The kit can be used for cells cultured in 96-well plates and is compatible with high-throughput studies. Preparation of templates for real-time PCR from cultured cells takes approximately 10 minutes and complete gene expression analysis (via One Step or Two Step qrt-pcr) is performed in approximately 2 hours. When used in combination with a reverse transcription kit, PrimeScript RT reagent Kit (Cat.# RR037A/B), cdna templates for real-time PCR can be synthesized in about 30 minutes, providing efficient high-throughput analysis. Templates can be prepared directly from small quantities of cells for analysis by high sensitivity qrt-pcr. Genomic DNA can be easily and efficiently removed using this kit. Excellent for detection of rare transcripts. Kit Components 3733 CellAmp Washing Buffer ml CellAmp Processing Buffer 10 ml DNase I for Direct RNA Prep 200 µl 5X Loading Buffer 2 1 ml 1M DTT (Dithiothreitol) μl ** For 200 wells of cultured cells in a 96-well plate. PCR PRODUCTS 1 A Comparison of Results between Western Blot Analysis and mrna Expression Real Time PCR Operational Flowchart 37

39 PrimerArray Series 1PCR PRODUCTS Real Time PCR See ordering information in the tables below Primer Sets for use in Analysis of Gene Expression Level by qrt-pcr Features Simultaneous Screening of 88 Types of Pathway Related Genes: using the difference in gene expression level Optimized for Best Performance with SYBR Premix Ex Taq II (Tli RNase H): no optimization necessary Easy Analysis: with a tool exclusive for use with PrimerArray products The PrimerArray series are primer sets for real time RT-PCR for analysis of the expression level of genes which relate to specific biological pathways. Having primers for 88 pathway related genes and 8 kinds of housekeeping genes, this product enables simultaneous screening of genes which have a difference in expression level by comparing control samples and unknown samples through a relative quantitative method. Each primer set has been confirmed to react well in real-time RT-PCR, and establish proper peaks in a melting curve analysis. The analysis tool, PrimerArray Analysis Tool Ver.1.0, may be downloaded from Takara s website ( For further detailed analysis of the genes which are confirmed to have difference in expression level, individual primers are available separately. Ordering Information (Human) Quanity Cat. # PrimerArray Cytokine-cytokine receptor interaction (Human) PrimerArray Cell cycle (Human) PrimerArray Cell adhesion molecules (Human) PrimerArray Jak-STAT signaling pathway (Human) PrimerArray Natural Killer cell mediated cytotoxicity (Human) PrimerArray Axon guidance (Human) PrimerArray Focal adhesion (Human) PrimerArray T cell receptor signaling pathway (Human) PrimerArray TGF-b signaling pathway (Human) PrimerArray Wnt signaling pathway (Human) 1 set (enough reagent for 12 plates) PH001 PH002 PH003 PH004 PH005 PH006 PH007 PH008 PH009 PH010 Ordering Information (Mouse ) Quanity Cat. # PrimerArray Cytokine-cytokine receptor interaction (Mouse) PrimerArray Cell cycle (Mouse) PrimerArray Cell adhesion molecules (Mouse) PrimerArray Jak-STAT signaling pathway (Mouse) PrimerArray Natural Killer cell mediated cytotoxicity (Mouse) PrimerArray Axon guidance (Mouse) PrimerArray Focal adhesion (Mouse) PrimerArray T cell receptor signaling pathway (Mouse) PrimerArray TGF-b signaling pathway (Mouse) PrimerArray Wnt signaling pathway (Mouse) 1 set (enough reagent for 12 plates) PN001 PN002 PN003 PN004 PN005 PN006 PN007 PN008 PN009 PN010 *A complete gene and primer list can be found on our website at

40 PCR Mycoplasma Detection Set PCR Mycoplasma Detection Set Cat.# reactions Rapid Detection, via Nested PCR, of the Mycoplasma Species Most Frequently Found to Contaminate Cell Cultures The PCR Mycoplasma Detection Set is a primer set de signed to detect the presence of mycoplasma in biological materials such as cultured cells. Nested PCR is performed first using primers hybridizing to highly conserved ribosomal genes (16S and 23S rrna), and then using primers hybridizing to the species-specific spac er region between these ribosomal genes. Verification of species is determined by gel electrophoresis of the amplified products. By performing PCR with this primer set, turn around time can be shortened from the conventional time of about one week to several hours. This kit al lows the high ly sen si tive and spe cif ic de tection of the fol low ing Mycoplasma spe cies: M. fermentans, M. hyorhinis, M. arginini, M. orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. neurolyticum, M. hypopneumoniae, M. capricolum and Ureaplasma urealyticum. Control Template In order to confirm that a sample does not include any inhibitory substances, a positive control experiment should be performed using the supplied control Template Set plus the sample template, and a reaction containing only sample template. In an inhibitor-free sample, bands derived from the Control Template (810 bp band with MCGp F1/R1 Primers, 590 bp with R2/R2 primers) appear in the positive control reaction; lack of bands in the control indicates the presence of an inhibitor. Note This product is optimized for use with TaKaRa Taq (not included) and may not perform as well with another company's Taq DNA Polymerase. Principle of PCR Mycoplasma Detection Set Set Components 6601 MCGp F1 Primer (20 pmol/µl) 50 µl MCGp R1 Primer (20 pmol/µl) 50 µl MCGp F2 Primer (20 pmol/µl) 50 µl MCGp R2 Primer (20 pmol/µl) 50 µl Control Template (1 ng/µl) 50 µl Product Amplification Size 1st PCR (bp) 2nd PCR (bp) M. hyopneumoniae M. neurolyticum M. fermentans M. pulmonis M. hyorhinis M. orale M. capricolum M. arthritidis M. salivarium M. hominis 370, , 148 M. arginini U. urealyticum 482, C Reference 1. Ueori, T. et al. (1992) System. Appl. Microbiol. 15: Related Products dntp Mixture (Cat.# 4030) TaKaRa Taq 250 units (Cat.# R001A) 1000 units (Cat.# R001B) TaKaRa Taq (with Mg 2+ (free) Buffer 250 units (Cat.# R001AM) 1000 units (Cat.# R001BM) 3000 units (Cat.# R001CM) TaKaRa Ex Taq 250 units (Cat.# RR001A) units (Cat.# RR001B) 3000 units (Cat.# R001C) (Mg 2+ free Buffer) 250 units (Cat.# RR001AM) (Mg 2+ free Buffer) 1000 units (Cat.# RR001BM) (Mg 2+ free Buffer) 3000 units (Cat.# RR001CM) PCR PRODUCTS 1 PCR Related Products Detection results with 11 species of Mycoplasma and 1 species of Ureaplasma using PCR: DNA was extracted from cultured Mycoplasma (11 species) and Ureaplasma (1 species) respectively. Nested PCR was performed in 100 μl using ~1 ng of extracted DNA, or 1 µg of human DNA and mouse DNA as negative control. In the 1st PCR, detection limits were 1 ng for M. capricolum(7), 100 pg for M. hyopneumoniae(1) and U. urealyticum(12), 10 pg for others. (Non-specific bands were detected when 100 ng human or mouse DNA was used as a template.) In the 2nd PCR, detection limits were 100 fg for M. capricolum (7) and M. hyopneumoniae (1), 10 fg for others. (Non-specific bands were not detected when 100 ng human or mouse DNA was used as a template.) 39

41 1PCR PRODUCTS O-157 (Verocytotoxin Genes) One-Shot PCR Screening Kit (Verocytotoxin Genes) One-Shot PCR Screening Kit Cat.# RR102A 48 tests Rapid Detection of Enterohemorrhagic E. coli (EHEC) Strains, Including O157:H7 Serotype, Via PCR Enterohemorrhagic E. coli (EHEC) strains, including the O157:H7 serotype, produce a verocytotoxin which causes hemorrhagic colitis in susceptible mammalian hosts. When detecting EHEC, it is important to quickly and accurately discriminate verocytotoxin-producing E. coli strains from nonpathogenic strains. Takara s O-157 One Shot PCR Screening Kit enables simple detection of EHEC strains by specifically amplifying verocytotoxin genes via PCR. One-Shot PCR solution contains all the reagents required to perform this PCR in a 0.2 ml PCR tube. The solution includes Ex Taq DNA Polymerase, which gives high yields, high efficiency, and allows highly sensitive detection in less time than conventional Taq Polymerase. All that needs to be added is the sample template DNA. Each tube also contains control template EC3 as a positive control. Because the size of the amplified fragments obtained from this control template are completely different from those obtained from the verocytotoxin gene (171 bp), the existence of verocytotoxin can be easily verified by gel electrophoresis. Kit Components RR102A 2X One-Shot PCR solution* µl (supplied in 0.2 ml PCR tube) * Contains: Primer EVC-1, 2, Control template EC3, TaKaRa Ex Taq DNA Polymerase, Ex Taq Buffer and dntps. 20 C References 1. Takao, T.et al. (1988) Microb. Pathog. 5: Jackson, M.P. et al. (1987)FES Microbio. Lett. 44: Ito, H.et al. (1990) Microb. Pathog. 8: Weinstein, D.L. et al. (1955)J. Bacteriol. 170: PCR Related Products O-157 (Verocytotoxin Genes) PCR Screening Set (Verocytotoxin Genes) PCR Screening Set Cat.# RR tests Detection of Genes of Verocytotoxin Type 1 and Type 2 by PCR from Enterohemorrhagic E. coli (EHEC) Strains, such as E. coli O157:H7 Takara s O-157 (Verocytotoxin Genes) PCR Screening Kit enables simple detection of the genes for Verocytotoxin Type 1 and Type 2 (171 bp) by PCR. This kit contains all the reagents required to perform this PCR. The solution includes TaKaRa Ex Taq which gives high yields, high efficiency, and allows highly sensitive detection in less time than conventional Taq Polymerase. All that needs to be added is sample template DNA. Kit Components RR100 EVC-1 primer (19 pmol/µl) 2 53 µl EVC-2 primer (19 pmol/µl) 2 53 µl TaKaRa Ex Taq (5 U/µL) 50 µl 10X Ex Taq buffer 1 ml dntp Mixture (ea. 2.5 mm) 800 µl Control Template EC3 (0.1 ng/µl) 10 µl 20 C References 1. Takao, T. et al. (1988) Microb. Pathog. 5: Jackson, M.P. et al. (1987) FEMS Microbio. Lett. 44: Ito, H. et al. (1990) Microb. Pathog. 8: Weinstein, D.L. et al. (1955) J. Bacteriol. 170:

42 O-157 (Verocytotoxin Genes) One-Shot PCR Typing Kit (Verocytotoxin Genes) One-Shot PCR Typing Kit Cat.# RR106A 48 tests Simple Detection of Both Type 1 and 2 Verocytotoxin Genes from EHEC This kit enables simple detection of Verocytotoxin genes from EHEC while simultaneously distinguishing between type 1 and type 2 Verocytotoxin genes in a single tube. The premixed solution (2X One Shot PCR solution in 0.2 ml tubes) contains all the reagents required to perform PCR including specific primers and TaKaRa Ex Taq. Detection can be initiated by simply adding a sample template DNA. Because this kit uses high efficiency TaKaRa Ex Taq, highly sensitive detection is possible in a shorter time than using regular Taq. Furthermore, a positive control template is supplied in each tube of this kit to prevent false negative detection. Because the size of the fragments amplified from this template (1070 bp) are easily distinguished from those obtained from Verocytotoxin gene, the existence of Verocytotoxin gene can be easily verified by gel electrophoresis. Kit Components RR106A 2X One-Shot PCR solution µl (supplied in 0.2 ml PCR tube) 20 C. Avoid repeated freeze-thaws. PCR PRODUCTS 1 O-157 (Verocytotoxin Genes) PCR Typing Kit (Verocytotoxin Genes) PCR Typing Kit Cat.# RR105A 100 tests Simple Detection of Both Type 1 and 2 Verocytotoxin Genes from EHEC This reagent set is designed for simple and quick detection of Verocytotoxin genes from Enterohemorrhagic E. coli (EHEC) while simultaneously making a distinction between type 1 and type 2 Verocytotoxin genes. Control Templates EC2 & EC3 These templates are utilized as the positive control templates to verify PCR amplification, using each of the primer pairs for the detection of Verocytotoxin genes from EHEC. Control template EC2 is designed to produce a 686 bp fragment with PCR primer EVT-1 & EVT-2. Control template EC3 is designed to produce a 686 bp fragment using PCR primer EVS-1 & EVS-2. Since the size of the amplified fragment from the control template differs from the amplified fragment derived from Verocytotoxin genes, the target fragment is easily discriminated even if a sample contamination has occurred. Kit Components RR105A EVT-1 primer* (19 pmol/µl) 53 µl EVT-2 primer* (19 pmol/µl) 53 µl EVS-1 primer* (19 pmol/µl) 53 µl EVS-2 primer* (19 pmol/µl) 53 µl TaKaRa Ex Taq (5 U/µL) 50 µl 10X Ex Taq buffer 1 ml dntp Mixture (ea. 2.5 mm) 800 µl Control Template EC2 (0.1 ng/µl) 10 µl Control Template EC3 (0.1 ng/µl) 10 µl *Manufactured by SHIMADZU Corporation. 20 C PCR Related Products 41

43 1PCR PRODUCTS PCR Related Products Multiplex PCR O-157/Verocytotoxin Genes Detection Kit Multiplex PCR 0-157/Verocytotoxin Genes Detection Kit Cat.# RR107A 50 tests Quick Detection of E. coli O-157 Verocytotoxin Genes Type 1 and 2 This reagent set is designed for simple and quick detection of Enterohemorrhagic E. coli O-157 gene (encoding the O-157 antigen protein) and Verocytotoxin genes from Enterohemorrhagic E. coli (EHEC). It also allows simultaneous typing of 0157 Types 1 and 2 Verocytotoxin genes in a single tube. Furthermore, false negative can be prevented by the use of the internal control template supplied in this kit as a positive control. Bacillus anthracis PCR Detection Kit Detection of Virulent Bacillus anthracis using PCR by Simultaneous Amplification of PA and CAP Genes The virulence of Anthrax (Bacillus anthracis) is due to the presence of two plasmids (px01 and px02). Plasmid X01 encodes three toxin components (PA: protective antigen, LF: lethal factor, EF: edema factor). Plasmid px02 encodes a group of genes involved in capsule synthesis (capa, capb, and capc ). Both of these plasmids must be present in the organism for virulence. This kit is designed to detect the PA gene contained in plasmid px01 and the capa gene in plasmid px02 via gel electrophoresis. This kit includes Takara s efficient Hot-Start PCR enzyme, TaKaRa Ex Taq HS, which results in very sensitive detection and reduced non-specific amplification due to mispriming or primer-dimers. The kit also contains an internal control for monitoring false negatives. Kit Components RR107A 10X Ex Taq Buffer 250 µl dntp Mixture (ea. 2.5 mm) 200 µl Primer Mixture* 500 µl Positive Control Template 500 µl TaKaRa Ex Taq (5U/µL) 12.5 µl *Manufactured by SHIMADZU Corporation. 20 C. Avoid repeated freeze-thaws. After initial thaw, aliquot and store at 20 C. Bacillus anthracis PCR Detection Kit Cat.# RR tests Kit Components RR027 TaKaRa Ex Taq HS (5 U/µL) 12.5 µl 5X Reaction Mixture* 500 µl (96 reactions) PA Primers (PA7, PA6) (10 µm each) 200 µl CAP Primers (M011, M012) (10 µm each) 200 µl 100 bp DNA Ladder (650 ng/5 µl) 50 µl 6X Loading Buffer** 60 µl * Includes dntp Mixture and Internal Controls. ** 36% Glycerol, 30 mm EDTA, 0.06% Bromophenol Blue, 0.06% Xylene cyanol. NOTE: This kit is designed as a rapid preliminary assay. For definitive assessment, this test should be combined with a general microbial test, such as gram staining. 20 C 42

44 Bacteria Screening PCR Kit Bacteria Screening PCR Kit Cat.# RR114A 50 reactions Detect Strains of Bacteria in the Enterobacteriaceae Family via PCR Kit Components RR114A Premix Solution* (2X conc.) 100 tubes 250 µl (100 reactions) Primer Mix ENT (5 µm each) 125 µl (50 reactions) The Bacterial Screening PCR Kit is designed to use the 16S rrna gene as a target Primer Mix BS (5 µm each) 125 µl (50 reactions) and employs an ENT primer for detecting strains in the Enterobacteriaceae family, Positive Control ENT 25 µl (10 reactions) including E. coli and Salmonella bacteria and the BS primer for detecting genus Positive Control BS 25 µl (10 reactions) Bacillus strains, (including Bacillus cereus), and the genus Staphylococcus, (including dh 2 O 3 tubes 1 ml Staphylococcus aureus strains). When used in combination with enrichment culture, 10% Chelex Solution** 2 vials 12 ml the Bacteria Screening PCR Kit can be used to rapidly detect the presence of even * Contents of Premix Solution: TaKaRa Ex Taq HS (0.1 U/μL), Optimized Buffer, (Mg 2+ minute numbers of bacteria in samples. Concentration: 4 mm), dntp Mixture (0.4 mm each) ** Chelex is a registered trademark of Bio-Rad Laboratories. Store the 10% Chelex solution at 4 C. Store all other components at 20 C. PCR PRODUCTS b-globin (human) Primer Set (Control Primer for PCR) b-globin (human) Primer Set Cat.# vials 500 pmol Amplified Fragment (bp) Amplify the Genes Corresponding to β-globin Region of Human Genomic DNA Primers can be used as the Positive Control for Confirming the Acceptability of the Extracted DNA to Template Primer Sequences PC03 (forward) d(acacaactgtgttcactagc) PC04 (reverse) d(caacttcatccacgttcacc) GH20 (forward) d(gaagagccaaggacaggtac) GH21 (reverse) d(ggaaaatagaccaataggcag) KM29 (forward) d(ggttggccaatctactcccagg) KM38 (reverse) d(tggtctccttaaacctgtcttg) Form Lyophilized (Reconstitute with by sterilized distilled water or TE buffer) 20 C reverse forward PC04 GH21 KM38 PC GH KM Quality Purity confirmed by HPLC Purity primer activity confirmed by PCR Note Primer PC03 has the same sequence as described in the reference below. However, some templates may not be amplified efficiently using this primer. In this situation, use the other primer pair. Reference 1. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Hom, G.T., Mullis, K.B. and Erlich, H.A. (1998) Science 239, PCR Related Products 43

45 1PCR PRODUCTS PCR Related Products Campylobacter (cdt gene) PCR Detection and Typing Kit Campylobacter (cdt gene) PCR Detection and Typing Kit Cat.# RR134A 100 reactions Detects Pathogenic Strains of Campylobacter The incidence of food poisoning caused by Campylobacter has become more prevalent. Campylobacter is a gram-negative, microaerophilic, spiral-shaped, non-spore forming bacteria. There are three species in the Campylobacter genus: Campylobacter jejuni (C. jejuni), Campylobacter coli (C. coli) and Campylobacter fetus (C. fetus). It is one of the foremost causes of human gastroenteritis in the world. Approximately 90% of the pathogenic bacteria for the gastroenteritis are C. jejuni and a small percent are C. coli. Campylobacter fetus (C. fetus) is isolated from septicemic or meningitidis specimens, and are considered pathogenic bacteria for these illnesses. The symptoms are different depending on the Campylobacter species, therefore, it is very important not only to detect the presence of the bacteria, but also to identify the species of the bacterium. Currently, detection and identification of Campylobacter is done by the culture method. This method is laborious due to the slow growth of the bacteria, which takes at least 2 days for pre-culture and then several days for identification of the species type. Therefore, a week is required for definite identification of the Campylobacter species. A rapid detection and identification method for Campylobacter using PCR has been developed by Takara Bio, Inc. This PCR detection and typing kit is designed to specifically and rapidly detect and identify the 3 types of Campylobacter species. The kit uses PCR to detect targets of the B subunit gene (cdtb gene) or C subunit gene (cdtc gene) of the Cytolethal distending toxin (cdt genes). In the Campylobacter species, there are occasionally strains which have deletions or mutations on their cdtb gene or cdtc gene. In order to increase the detection accuracy of this kit, detection of both the cdtb and cdtc genes is performed. The kit uses Takara s high performance enzyme, Ex Taq Hot Start Version, which allows high sensitivity detection of the target genes. Takara s Ex Taq HS decreases mis-priming during preparation of reaction solutions and non-specific amplifications derived from primer dimers. This kit was designed by TaKaRa through the cooperation with Osaka Prefecture University and Fuso Pharmaceutical Industries, Ltd. Kit Components RR134A* Premix Ex Taq HS** (2X conc.) µl cdtb Primer Mix 250 µl cdtc Primer Mix 250 µl dh 2 O 3 1 ml C. jejuni Positive Control 150 µl C. coli Positive Control 150 µl C. fetus Positive Control 150 µl * 50 reactions for cdtb gene detection and 50 reactions for cdtc gene detection. ** 2X concentration of PCR reaction reagent which contains TaKaRa Ex Taq HS, Reaction Buffer, and dntp mixture. Avoid repeated freeze-thaws; upon first thaw, aliquot 25 µl into separate tubes and store at 20 C. 20 C References 1. Worada Samosornsuk, et al. (2007) Microbiol. Immunol., 51 (9), Asakura, M., et al. (2007) Microb. Pathog., 42,

46 PCR Human Papillomavirus Detection Set PCR Human Papillomavirus Detection Set Cat.# reactions Detection of Human Papillomavirus PCR Human Papillomavirus Detection Set is a primer set designed to detect the presence of human papillomavirus (HPV) 16, 18, and 33 which are often found in cervical carcinoma. This primer set allows simple and sensitive detection of HPV 16, 18 or 33 by specific amplification of their DNA using PCR. Included in this set are the specific primers for amplification of the sequence containing E6 region of HPV 16, 18 and 33 (140 bp for HPV 16, 18 and 141 bp for HPV 33) and the HPV type-specific probes for the subsequent hybridization. This primer set will work most efficiently when used in conjunction with TaKaRa Taq (Cat.# R001). Set Components 6602 HPVpF (common forward primer, 25 pmol/µl) 100 µl HPVp16R (HPV 16 reverse primer, 25 pmol/µl) 50 µl HPVp18R (HPV 18 reverse primer, 25 pmol/µl) 50 µl HPVp33R (HPV 33 reverse primer, 25 pmol/µl) 50 µl HPVb16 (HPV 16 probe, 25 pmol/µl) 10 µl HPVb18 (HPV 18 probe, 25 pmol/µl) 10 µl HPVb 33 (HPV 33 probe, 25 pmol/µl) 10 µl Control Template HPVT16 (HPV16, 1 ng/µl) 50 µl HPVT18 (HPV18, 1 ng/µl) 50 µl HPVT33 (HPV33, 1 ng/µl) 50 µl 20 C Application Example Amplification and detection of HPV16 DNA by dot hybridization can be performed. A: Agarose gel electrophoresis of amplified DNA B: Detection of amplified DNA by dot hybridization using 5 -end [ 32 P] labeled HPVb16 probe. PCR PRODUCTS 1 Principle of the Amplified Region Control Template This set includes Control Template to verify the PCR amplified DNA fragments. Amplification using HPVT16, HPVT18, HPVT33 as template and primer pairs of HPVpF/HPVp16R, HPVpF/HPVp18R, HPVpF/HPVp33R respectively yields 70 bp of DNA fragments. As this Control Template contains the sequences complementary to the probes provided in the set, it can be used as positive control for each type of HPV in dot hybridization. PCR Related Products Amplification and Detection of HPV16 DNA by Gel Analysis and Dot Hybridization 45

47 1PCR PRODUCTS PCR Human Papillomavirus Typing Set PCR Human Papillomavirus Typing Set Cat.# reactions Identify a Broad Range of Human Papillomavirus Types PCR Human Papillomavirus Typing Set, a primer set for PCR, is designed to identify a broad range of human papillomavirus (HPV) types. This set utilizes two pairs of consensus primers, designed for amplifying the sequence of HPV homologous region containing the E6 and E7 regions of HPV ( bp). Of the HPVs, malignant HPV- 16, -18, -33, -52b and -58 are amplified using the HPVpU-1M/HPVpU-2R primer pair; and benign HPV-6 and -11 are amplified using the HPVpU-31B/HPVpU-2R primer pair. With digestion of PCR products by restriction enzymes (Acc I, Afa I, Ava I, Ava II and Bgl II, available as Enzyme Set A (Cat.# 6604) and subsequent agarose gel electrophoresis, HPV types can be identified. This primer set is optimized for use with TaKaRa Taq (Cat.# R001)*. *TaKaRa Taq (R001) is not supplied with this set. Set Components 6603 HPVpU-1M (malignant forward primer, 25 pmol/µl) 50 µl HPVpU-31B (benign forward primer, 25 pmol/µl) 50 µl HPVpU-2R (common reverse primer, 25 pmol/µl) 100 µl Control Template HPV-TM (malignant, 1 ng/µl) 50 µl Control Template HPV-TB (benign, 1 ng/µl) 50 µl 20 C Control Template This set includes both malignant and benign control template to verify the PCR amplified DNA fragment. Amplification using HPV-TM, HPV-TB as template and primer pairs of HPVpU-1M / HPVpU-2R, HPVpU-31B / HPVpU-2R respectively yields approximately 60 bp of DNA fragments which can be distinguished from the one amplified from HPV. PCR Related Products Principle of the Amplified Region. Fragment sizes obtained after restriction enzyme digestion. Application Example: Identification of Malignant Types Gel electrophoresis after digestion of amplified DNA by Ava II indicates the following malignant types. The amplified fragment of HPV16 (238 bp) was cut into 157 bp and 81 bp; HPV18 (268 bp) into 172 bp and 96 bp; and, HPV33 (244 bp) into 136 bp and 108 bp. The amplified fragments of HPV31, 52b and 58 were not digested by Ava II. Enzyme Set A for the HPV Typing Set Enzyme Set A Cat.# reactions Set Components Enzyme Set A Acc I U 6604 Afa I U This enzyme set is designed for use with the HPV Typing Set (Cat.# 6603). It includes Ava I U Ava II U the restriction enzymes Acc I, Afa I, Ava I, Ava II, and Bgl II. Bgl II U Related Products PCR Human Papillomavirus Typing Set (Cat.# 6603) 46

48 Bacterial Pathogens PCR Positive Control Templates Positive Control Amplified Positive Amplified Target Template Cat.# Specific Primers Control DNA DNA Fragment VP1 S031 VPD-1 & VPD-2 (S001) 688 bp 251 bp VPS-1 & VPS-2 (S002) 688 bp 210 bp VP2 S046 VPR-1 & VPR-2 (S028) 666 bp 250 bp EC1 S032 ELT-1 & ELT-2 (S003) 690 bp 263 bp ESH-1 & ESH-2 (S004) 691 bp 131 bp EC2 S033 ESP-1 & ESP-2 (S005) 689 bp 123 bp EVT-1 & EVT-2 (S006) 686 bp 349 bp EC3 S034 EVS-1 & EVS-2 (S007) 686 bp 404 bp EVC-1 & EVC-2 (S008) 695 bp 171 bp SE1 S035 SEA-1 & SEA-2 (S009) 695 bp 423 bp SEB-1 & SEB-2 (S010) 694 bp 391 bp SE2 S036 SEZ-1 & SEZ-2 (S011) 697 bp 146 bp SED-1 & SED-2 (S012) 695 bp 499 bp ST S037 SEE-1 & SEE-2 (S013) 695 bp 557 bp TST-1 & TST-2 (S015) 694 bp 228 bp SS S038 INV-1 & INV-2 (S016) 691 bp 293 bp IPA-1 & IPA-2 (S017) 689 bp 242 bp VC S039 VCT-1 & VCT-2 (S014) 670 bp 307 bp SN S040 SIN-1 & SIN-2 (S018) 689 bp 378 bp STN-1 & STN-2 (S019) 690 bp 264 bp CP S041 CPE-1 & CPE-2 (S020) 667 bp 456 bp BS1 S042 BAS-1 & BAS-2 (S021) 691 bp 284 bp BBS-1 & BBS-2 (S022) 691 bp 314 bp BS2 S043 BCS-1 & BCS-2 (S023) 690 bp 290 bp BDS-1 & BDS-2 (S024) 690 bp 497 bp BS3 S044 BES-1 & BES-2 (S025) 691 bp 266 bp BFS-1 & BFS-2 (S026) 691 bp 332 bp BS4 S045 BGS-1 & BGS-2 (S027) 668 bp 488 bp Highly Specific Detection of Bacterial Pathogens using PCR Each set of the Bacterial Pathogens PCR Positive Control DNA can be used to verify the performance of PCR amplification when using the Bacterial Pathogenic Primer Sets (Cat.# S001-S028). The DNA fragment sizes amplified from these controls are different from those amplified from the bacterial genome, so contamination of the control can be easily monitored. Quantity 5 ng Form 100 pg/µl in sterile TE buffer Volume 50 µl 20 C PCR PRODUCTS 1 PCR Related Products Related Products Bacterial Pathogens PCR Primers (Cat.# S001 S028) 47

49 Bacterial Pathogens PCR Primer Sets 1PCR PRODUCTS Amplified Primer Sets Cat.# Detectable Gene Fragment VPD-1 & VPD-2 S001 Thermostable direct hemolysin (tdh) gene of Vibrio parahaemolyticus 251 bp VPS-1 & VPS-2 S002 Thermostable direct hemolysin-related (trh1) gene of Vibrio parahaemolyticus 210 bp ELT-1 & ELT-2 S003 Gene of the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) 263 bp ESH-1 & ESH-2 S004 Gene of the heat-stable enterotoxin for human (STh) of ETEC 131 bp ESP-1 & ESP-2 S005 Gene of the heat-stable enterotoxin for porcine (STp) of ETEC 123 bp EVT-1 & EVT-2 S006 Gene of the Verocytotoxin type 1 (VT1; Shiga-like toxin type 1) of ETEC, or Verocytotoxin-producing E. coli; (VTEC) 349 bp EVS-1 & EVS-2 S007 Gene of the Verocytotoxin type 2 (VT2 VT2vha, VT2vhb, VT2vp1; Shiga-like toxin type II) of EHEC, VTEC 404 bp EVC-1 & EVC-2 S008 Gene of the Verocytotoxin type 1 and type 2 (VT1, VT2 VT2vha, VT2vhb, VT2vp1; Shiga-like toxin type I & II) of EHEC, or VTEC 171 bp SEA-1 & SEA-2 S009 sea (enta) gene encoding staphylococcal enterotoxin A 423 bp SEB-1 & SEB-2 S010 seb (ent B) gene encoding staphylococcal enterotoxin B 391 bp SEZ-1 & SEZ-2 S011 sec (ent C) gene encoding staphylococcal enterotoxin C 146 bp PCR Related Products SED-1 & SED-2 S012 sed (entd) gene encoding staphylococcal enterotoxin D 499 bp SEE-1 & SEE-2 S013 see (ent E) gene encoding staphylococcal enterotoxin E 557 bp VCT-1 & VCT-2 S014 ctx gene encoding Vibrio cholera enterotoxin 307 bp TST-1 & TST-2 S015 tst gene encoding toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus 228 bp INV-1 & INV-2 S016 inve gene of Shigella or enteroinvasive E.coli 293 bp IPA-1 & IPA-2 S017 ipah gene of Shigella or enteroinvasive E.coli 242 bp SIN-1 & SIN-2 S018 inva gene of Salmonella sp. 378 bp STN-1 & STN-2 S019 Enterotoxin gene of Salmonella sp. 264 bp CPE-1 & CPE-2 S020 Enterotoxin gene of Clostridium perfringens 456 bp BAS-1 & BAS-2 S021 Type A neurotoxin gene of Clostridium botulinum 284 bp BBS-1 & BBS-2 S022 Type B neurotoxin gene of Clostridium botulinum 314 bp BCS-1 & BCS-2 S023 Type C neurotoxin gene of Clostridium botulinum 290 bp BDS-1 & BDS-2 S024 Type D neurotoxin gene of Clostridium botulinum 497 bp BES-1 & BES-2 S025 Type E neurotoxin gene of Clostridium botulinum 266 bp BFS-1 & BFS-2 S026 Type F neurotoxin gene of Clostridium botulinum 332 bp BGS-1 & BGS-2 S027 Type G neurotoxin gene of Clostridium botulinum 488 bp VPR-1 & VPR-2 S028 Thermostable direct hemolysin-related hemolysis gene of 250 bp Vibrio parahaemolyticus Abbreviations: E. coli: Escherichia coli, ETEC: enterotoxigenic Escherichia coli, VTEC: verocytotoxin-producing E. coli, staphylococcal: Staphylococcus aureus Note: Primers are optimized for use with TaKaRa Taq or Ex Taq DNA Polymerase (not included). Highly Specific Detection of Bacterial Pathogens using PCR These primer sets are designed to specifically detect bacterial pathogens in food using PCR. Band sizes are listed in the table above. Quantity 1000 pmol Form 19 pmol/µl in sterile water Volume 53 µl Related Products Positive Control Templates for PCR Pathogen Detection (Cat.# S031 S045) Note: Primers S001 S020 are manufactured by Shimadzu Corporation 48

50 Cloning, Mapping & cdna Synthesis DNA Ligation Kits, Versions 1.0 and DNA Ligation Kit LONG...52 DNA Ligation Kit, Mighty Mix...53 DNA Blunting Kit...54 Reagent Set for Mighty Cloning Kit (Blunt End) Cassettes & Cassette Primers...54 Reagent Set for Mighty Cloning Kit (Blunt End) Cassettes & Cassette Primers...55 pkf3 DNA (Enforcement Cloning Vector)...55 COMPETENT CELLS E. coli Competent Cells...56 E. coli Electro-Competent Cells...56 GEne Transfer and Expression RetroNectin Reagent and RetroNectin Precoated Dishes pdon-ai-2 DNA...58 pmei-5 DNA...58 pbapo-cmv Vector series NEW...58 pbapo-cmv Vector series NEW...59 Adenovirus Expression Vector Kit, Adenovirus Genome DNA-TPC and Adenovirus Dual Expression Kit (EF1a) NEW. 59 Yeast Transformation System Aureobasidin A...63 Aureobasidin A Vectors...60 paur Primers...63 Pyrithiamine Vectors...63 molecular biology ssrna Ladder Marker...75 Protein Molecular Weight Marker...75 Ribonucleoside 5'-Triphosphates...76 Deoxyribonucleoside 5 -Triphosphates...76 dntp Mixture...76 M13 Primers...77 M13 Primer RV-N...77 Ladderman (BcaBEST ) Sequencing Primers...77 Random Primers Adaptors...78 Phosphorylated Linkers Nonphosphorylated Linkers...79 Synthetic sirna Quantitation Core Kit NEW...80 DNA Labeling and Detection Kits Random Primer DNA Labeling Kit, Ver Ladderman DNA Labeling Kit...81 cdna SYNTHESIS PrimeScript Reverse Transcriptase...80 PrimeScript 1st Strand cdna Synthesis Kit...81 Sequencing, Mutagenesis and Mutation Analysis Comparison of Takara s Site-Directed Mutagenesis Systems...83 Deletion Kit for Kilo-Sequencing...84 RNase-Free Water...84 MOLECULAR BIOLOGY 2 Table of Contents RNA/Protein/DNA Extraction Dr. GenTLE Systems (Gene Trapping by Liquid Extraction)...64 Protein and RNA Extraction Kit for Mammalian Cells (PAREx Kit)...65 Yeast Processing Reagent (for total RNA preparation)...65 Miniprep DNA purification Set...65 TaKaRa DEXPAT (DNA Extraction from Paraffinembedded Tissue)...66 TaKaRa DEXPAT Easy NEW...66 TaKaRa RECOCHIP...67 SUPREC -01 DNA Purification Cartridge...67 Plant Products Fruit-mate for RNA Purification...68 High-Salt Solution for Precipitation (Plant) NEW...68 Plant DNA Isolation Kit NEW...68 Agrobacterium tumefaciens LBA4404 Electro-Cells...69 pri 909/910 DNA...70 pri 101 DNA Vectors...70 pri 201 DNA Series NEW...70 Nucleic Acids Takara DNA Vectors and Genomic DNAs...71 DNA Molecular Weight Standard Markers...72 molecular biology reagents DNA-OFF...84 RNase-OFF...84 Modifying enzymes Summary of TaKaRa Modifying Enzymes...85 AMV Reverse Transcriptase XL...86 Terminal Deoxynucleotidyl Transferase...86 DNA Topoisomerase I...86 Phosphatases...87 Ligases...88 Polymerases...89 Nucleases...92 Micrococcal Nuclease...92 Cryonase Cold-Active Nuclease...93 Ribonuclease Inhibitors...95 Restriction Enzymes...94 Others IPTG (dioxane free) (Isopropyl-b-Dthiogalactopyranoside) X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-Galactoside) Proteinase K Westase Enzyme Agarose L

51 2MOLECULAR BIOLOGY Cloning, Mapping & cdna Synthesis DNA Ligation Kits, Versions 1.0 and 2.1 DNA Ligation Kit, Ver. 1.0 Cat.# reactions DNA Ligation Kit, Ver. 2.1 Cat.# reactions High-Efficiency DNA Ligation in as Little as 3 Minutes (30 Minutes for Some ) Suitable for l DNA Concatenation (Version 1) Ligation in Small Reaction Volumes (Version 2.1) Takara s DNA Ligation Kits are simple, two-component systems that allow rapid performance of all cloning procedures requiring the covalent joining of DNA molecules in vitro. Using an optimized buffer system and T4 DNA Ligase, these kits allow ligation in 30 minutes with high transformation efficiency. For general ligation reactions, these kits allow good efficiency in only 3 minutes. The reactions can be used directly in some bacterial transformations without DNA purification. The kits have similar, simple procedures and give nearly equivalent transformation efficiencies. However, Version 2.1 contains a convenient premix of enzyme and buffer that further minimizes pipetting steps and final reaction volume in standard reactions. Solution II of Version 2.1 is designed for linker addition ligations. Transformation efficiency can be improved by the addition of Solution III (Transformation Enhancer) into circular ligation reactions before transformation. Version 1.0 can also be used for DNA concatenation using Solution B alone Kit Com po nents 6021 Version 1 Solution A: Reaction Buffer 3 1 ml Solution B: Enzyme Solution µl 6022 Version 2.1 Solution I: Enzyme Solution µl (contains ligase and reaction buffer) Solution II: Concatenation Buffer µl Solution III: Transformation Enhancer µl efficiency DNA) g Transformation ( transformants/ µ Application Ligation for generating circular DNA Insertion of DNA fragments into plasmid vectors Insertion of PCR fragments into T-Vector Insertion of linker DNA into plasmid vectors Self-circularization Ligation for generating linear DNA Adaptor, linker ligation to cdna Insertion of DNA fragments into phage vectors* *Version 1 is recommended Reference 1. Hayashi, K. et al. (1986) Nucleic Acids Res. 14: Version 1 (Volume ratio) DNA Solution (1) Solution A (4-8) Solution B (1) DNA Solution I (1) Solution B (1) (300 mm NaCl) Version 2.1 (Volume ratio) DNA Solution (1) Solution (1) DNA Solution (1) Solution I (2) Solution II (1) 16 C, 30 min. 25 C, 3 min. Lane 1: λ-hind III digest 2: λgt11/ecor I arms 3: Control insert DNA 4: Reactant of DNA Ligation Kit Ver. 1 5: Reactant of DNA Ligation Kit Ver Ver. 1 Ver. 2.1 Three Minute Ligation of a DNA Fragment into a Phage Vector. Control insert DNA (plasmid vector) digested with EcoR I was ligated with to λgt11/ecor I arms, dephosphorylated, for 3 minutes at 25 C. Efficiency of Linker Ligation. A pbgl II linker d[pcagaatctg] was ligated with dephosphorylated Hinc II puc118 DNA. 10 ng of the ligation reaction was transformed into E. coli JM109 Competent Cells ( transformants/µg DNA), and the transformation efficiency was calculated from the yield of white colonies. 50

52 7 Application: Using DNA Ligation Kits, Vers. 1.0 & 2.1 efficiency DNA) g Transformation ( transformants/ µ C, 30 min. 25 C, 3 min. Ligation Kit 16 C, 30 minutes Conventional Method 16 C, 16 hours MOLECULAR BIOLOGY 10 1 Ver. 1 Ver. 2.1 (Blunt-end) Ver. 1 Ver. 2.1 (Sticky-end) Comparison Between the Two DNA Ligation Kits. puc118 DNA was digested with Hinc II (blunt ends) or EcoR I (sticky ends), and aliquots were self-ligated following the protocol for the DNA Ligation Kit, Version 1 and Version 2.1, using a 3-minute ligation at 25 C, or a 30-minute ligation at 16 C. Aliquots of each reaction were used to transform competent JM109 cells, and the results were compared after calculating transformation efficiency. T ransformation efficiency (10 cfu/ µg λ gt 11 DNA ) DNA Ligation Kit Ver.1 T4 DNA ligase min. 16 hr. Ligation reaction time Efficiency of Cloning into Plasmid Vectors using the DNA Ligation Kit (Version 2.1). Fifty nanograms of EcoR I-digested and dephosphorylated puc118 DNA were ligated to a 1.5 kb EcoR I insert at various insert to vector molar ratios following the protocol supplied with the DNA Ligation Kit (Version 2.1). The same fragments were ligated using conventional T4 DNA ligase reactions. Aliquots of the ligation mixtures were used to transform JM109 competent cells and the results were compared after calculating transformation efficiency. T ransformation efficiency (10 5 cfu/ µ g DNA ) Insert(+) : 16 C, 30 min. : 25 C, 5 min Insert(-) 2 Cloning, Mapping & cdna Synthesis Ligation of l DNA using the DNA Ligation Kit (Version 1). pbr322 DNA digested with EcoR I was ligated to dephosphorylated lgt11 EcoR I arms using the DNA Ligation Kit (Version 1). The same reaction was performed with T4 DNA ligase. The ligated DNA was packaged using a commercially available packaging system, and the resulting recombinant phage were assayed by infection of Y1090 cells. TA Cloning Efficiency of the DNA Ligation Kit, Version 2.1. The DNA Ligation Kit Version 2.1 offers excellent transformation efficiency in cloning when incubated at 16 C for 30 minutes. Even using the quick protocol (25 C for 5 minutes), adequate efficiency was obtained for routine cloning applications. 51

53 2MOLECULAR BIOLOGY DNA Ligation Kit LONG DNA Ligation Kit LONG Cat.# reactions Vector Construction with Long DNA Fragments BAC Library Construction Cloning of Long cdna Fragments Features Superior Ligation Efficiencies: Especially for long DNA fragments Simple Protocol Long Ligation Control: Vector and insert provided Takara s DNA ligation Kit LONG is a powerful tool for cloning DNA fragments over 10 kb in length. The kit contains an optimized ligase/buffer system which enables ligation of long fragments without difficult techniques and special expertise. It is especially well-suited for the construction of BAC libraries. Figure 1 shows a comparison of the ligation efficiencies several commercially available ligation kits with Takara s DNA Ligation Kit LONG for the ligation of DNA fragments of varying sizes (2 18 kb). Takara s LONG Kit gave efficiences of about 50 times higher than the other commercially available DNA ligation kits for ligation of high molecular weight DNA (18 kb) into a cloning vector. The DNA Ligation Kit LONG provided superior performance not only with high molecular weight DNA, but also with smaller-size DNA inserts. Kit Components 6024 DNA Ligase <LONG> 50 µl 10X LONG Ligation Buffer 300 µl Control Insert DNA/Hind III (18 kb) 50 µl Control Vector (puc 8/Hind III/BAP) 10 µl dh 2 O 1 ml * For cohesive-end ligation, the kit provides 50 reactions. For blunt-end ligation, the kit supplies reagents for 10 reactions % 110.0% 80.0% 60.0% 40.0% 20.0% Ligation Time Course (Cohesive end) Cloning, Mapping & cdna Synthesis Transformants x 10 6 / µg kb 0.0% 4kb 10kb 18kb 15min 30min 1 hr 3 hr 6 hr 15 hr Time Course of Cohesive-end Ligation 8 kb DNA Fragment Digested with Hind III Ligated into the puc 8/ Hind III/BAP Vector Following the Standard Protocol. Ligation products were transformed into chemically competent E. coli cells and grown overnight on LB-amp plates at 37 C. Lig LONG T4 Ligase Mighty Mix Company A Company B Company C Company D Insert DNA size Figure 1. Comparison of Ligation Efficiency with Various DNA Ligation Kits. Several size DNA fragments, (2kb, 4kb, 10kb and 18kb) digested by Hind III were ligated into the cloning vector puc118/hind III/BAP using Ligation Kit Long (Lig LONG) or other commercially available DNA ligation kits. Ligation products were transformed into E.coli DH5α cells and grown overnight on LB-amp plates at 37 C. 52

54 DNA Ligation Kit, Mighty Mix DNA Ligation Kit, Mighty Mix Cat.# kit (100 reactions) High-efficiency DNA Ligation in as Little as 5 Minutes (30 Minutes for Some ) Ligation in Small Reaction Volumes TA and Blunt End Cloning Features High Efficiency: Facilitates high-efficiency ligations for cohesive, blunt-end, and TA cloning Convenient: One solution premix eliminates pipetting steps, and the reaction mixture can be used directly in transformations without purification Fast: Reactions can be incubated at 30 minutes at 16 C or 5 minutes at 25 C, depending on DNA ends used Small Volumes: Ten microliter reactions possible Takara s DNA Ligation Kit, Mighty Mix, is a one-solution premix ligation reagent that offers high efficiency ligations, particularly for blunt-ended ligation. The reaction can be performed for 5 minutes at 25 C (rapid protocol), or 30 minutes at 16 C (standard protocol) with equal results for both circular and linear DNA ligations. The 2X Ligation Mix allows small ligation reaction volumes (10 µl) when DNA is limiting. The kit includes sufficient reagent for 100 ligation reactions. Ligation products reaction can be used directly in bacterial transformation. Colonies/g Vector 1.0 X X X X X X X X X X 10 6 White Colonies Blue Colonies Kit Com po nents 6023 Ligation Mix µl * Kit components support 100 reactions when 7.5 µl of Ligation Mix is used per reaction. Reaction Conditions 0.0 X X 10 0 Mighty Mix Company A Company B Company C Mighty Mix Company A Company B Company C Colonies/g Vector 1.4 X X X X X X X 10 5 DNA Solution: Ligation Mix Ratio (Volume) White Colonies Blue Colonies Reaction Condition Insertion Standard Protocol 1:1 16 C, 30 min. of DNA 1:1 25 C, 5 min. Fragments into Plasmid Rapid Protocol Vectors Cloning of PCR Fragments into 1:1 16 C, 30 min. T-Vector Ligation Generating Linear DNA (self circularization) 1:1 16 C, 30 min. Linker or Adaptor Ligation Insertion of Linker DNA into Plasmid Vector Linker or Adaptor Ligation into cdna Insertion of DNA Fragments into l-phage vectors (in vitro packaging) 1:1 16 C, 30 min. 1:2 16 C, 30 min. 1:2 26 C, 10 min. Note: This kit retains activity through 50 freeze-thaw cycles. The Ligation Mix should be thawed on ice (5 10 minutes) and mixed well by pipette prior to use. Reference 1. Hayashi, K. et al. (1986) Nucleic Acids Res. 14: MOLECULAR BIOLOGY 2 Cloning, Mapping & cdna Synthesis Blunt-Ended Ligation Efficiency Comparison with Takara s DNA Ligation Kit, Mighty Mix and Three Competitors. A: Company P, B: Company B, and C: Company N. Each ligation reaction contained 25 fmol of BAP-treated puc118-hinc II vector and 75 fmol of a 500 bp insert. TA-Vector Ligation Efficiency Comparison of Takara s DNA Ligation Kit, Mighty Mix Against Three Competitors: A: Company P, B: Company B, and C: Company N. Each ligation reaction contained 13 fmol of a pt7 Blue T-Vector and 39 fmol of a 2.08 kb PCR product. 53

55 2MOLECULAR BIOLOGY Cloning, Mapping & cdna Synthesis DNA Blunting Kit DNA Blunting Kit Cat.# reactions Quick and Easy Blunting of DNA Fragment Ends The DNA Blunting Kit allows the conversion of 3 and 5 over hangs to blunt ends. This conversion is ac com plished by the 3 g5 exonuclease and 5 g3 poly merase activities of T4 DNA Polymerase. The resulting blunt-ended DNA can be ligated efficiently into a vector using the same optimized buffer system used in Takara s DNA Ligation Kits, then used directly in bacterial transformation or in vitro packaging procedures without DNA purification. Reagent Set for Mighty Cloning Kit (Blunt End) Quick and Easy Preparation of PCR Products for Cloning into Blunt-end Vectors Takara s Reagent Set for Mighty Cloning Kit (Blunt End) is designed to simply and quickly prepare PCR products for cloning into blunt-ended vectors. Because the blunting and 5 -phosphorylation reactions are done simultaneously, both blunt- and -da ended products can be prepared in a single reaction. This kit eliminates the need for pre-treatment (such as enzyme inactivation, removal of unused dntps and primers, etc.), of PCR products. This kit includes the same ligation reagents as are included in Takara s DNA Ligation Kit (Mighty Mix), which provide simple, quick, and efficient ligation. The kit is suitable for blunt-end cloning of PCR products obtained with PrimeSTAR HS and e2tak DNA Polymerases (not available in all regions). Kit Components 6025 T4 DNA Polymerase 20 µl 10X Buffer 30 µl DNA Dilution Buffer 500 µl Ligation Solution A µl Ligation Solution B 150 µl Reference 1. Sambrook J. et al. (1989) in Mo lec u lar Cloning, A Laboratory Manual, Cold Spring Har bor Lab o ra to ry. Reagent Set for Mighty Cloning Kit (Blunt End) Cat.# reactions Kit Components X Blunting Kination Buffer 40 µl Blunting Kination Enzyme Mix 20 µl Ligation Mighty Mix* 120 µl puc118-hinc II/BAP (50 ng/µl) 20 µl Control Insert** (200 ng/µl) 10 µl ddh 2 O 500 µl * Ligation Mighty Mix is the same reagent that is included in DNA Ligation Kit (Mighty Mix) (Cat.# 6023). ** 500 bp PCR fragment amplified with TaKaRa Ex Taq using l DNA as a template. 20 C Use with PrimeSTAR for easy cloning 54

56 I I Cassettes & Cassette Primers EcoR I Cassette Cat.# pmol (500 ng) Hind III Cassette Cat.# pmol (500 ng) Pst I Cassette Cat.# pmol (500 ng) Sal I Cassette Cat.# pmol (500 ng) Sau3A I Cassette Cat.# pmol (500 µg) Xba I Cassette Cat.# pmol (500 ng) Cassette Primer C1 Cat.# ,000 pmol Cassette Primer C2 Cat.# ,000 pmol Cloning of cdna or Genomic DNA using PCR Cassette Primer: used as a Primer for Sequencing An RNA Probe can be Prepared by in vitro Transcription using T7 Promoter within Cassette These products have the same sequences as the components of LA PCR in vitro Cloning Kit (Cat.# RR015). Form Lyophilized (Reconstitute with sterilized distilled water or TE buffer) Purity Confirmed by HPLC 20 C MOLECULAR BIOLOGY 2 pkf3 DNA (Enforcement Cloning Vector) pkf3 DNA Cat.# µg (0.2 OD) DNA Sequencing using pkf3 Primers pkf3 DNA is a vector which is used with enforcement cloning system. pkf3 DNA contains a chloramphenicol resistant gene and lethal gene rpsl (streptomycin-sensitive ribosomal protein) downstream of the tryptophan promoter. This vector includes a multi-cloning site which possesses an amber mutation and 39 restriction sites on the rpsl gene. Therefore, normal rpsl will not be expressed if a target gene is inserted into these restriction sites. Only clones with inserts will be propagated in trpr -, rpsl - and supe - host such as E. coli TH2, allowing easy selection of targeted clones. Concentration 500 µg/ml Vector Size 2,246 bp GenBank Entry Name: Accession No.: ECORPS12B D14641 Form 10 mm Tris-HCl (ph 8.0), 1mM EDTA 20 C I E c o T 14 I- ( D s a I ) P sh B I H pa I ( S p e I) N co - ( S p e I ) P vu I I I B a n I I N sp V - E a e I ) S ac - ApaL I A v a ) B al I ( X ho I ( Hgl E II 2.0 amber S ph I ori de N I M lu I - A c c ) B st I ( E co R I 0/2.247 POtrp A or 51H I pkf3 2,246 bp F sp I H in d I II pl S I rpsl 4a m + P I oo 6 I Bst -Ec 5 BspM I Nhe I 0.5 Sca I Ssp I Sse8387 I-Pst I Fba I * I o52 -( e I Not -Ec I Bgl I S tu I E a I) A v I) Sma I -( a BamHI Kpn I Sac I Xba I D s I) I -( a A cc III Ava II S al I-( A cc I) Cla I * P vu I Cloning, Mapping & cdna Synthesis 1.0 cat-5 (Spe I) 55

57 E. coli Competent Cells 2MOLECULAR BIOLOGY Cloning, Mapping & cdna Synthesis E. coli HB101 Competent Cells Cat.# x 100 µl E. coli JM109 Competent Cells Cat.# x 100 µl E. coli CJ236 Competent Cells Cat.# x 100 µl E. coli BMH71-18 muts Competent Cells Cat.# x 100 µl E. coli MV1184 Competent Cells Cat.# x 100 µl E. coli TH2 Competent Cells Cat.# x 100 µl E. coli DH5a Competent Cells* Cat.# x 100 µl E. coli HST02 Competent Cells Cat.# x 100 µl E. coli HST04 dam-/dcm- Competent Cells Cat.# x 100 µl Application Used for Heat Shock Transformation of DNA into E. coli E. coli cells treated with calcium ions can take in foreign DNA (i.e., plasmid or phage DNA) and are called competent cells. Competent cells are used in recombinant DNA experiments for transformation or transfection. Preparation of competent cells with high transformation efficiency can be difficult, but is especially important when the abundance of the target gene is low (i.e., gene library preparations, recombinant plasmid preparations or subcloning). Takara offers nine Competent Cell strains with guaranteed high transformation efficiency prepared by the modified Hanahan method 1. (E. coli HB101, JM109, CJ236, BMH71-18 mut S, MV1184, TH2, DH5α, HST02, HST04; all of these are EK1 host cells.) *E.coli DH5α competent cells are not available in the U.S. E. coli Electro-Cells E. coli HB101 Electro-Cells Cat.# x 50 µl E. coli JM109 Electro-Cells Cat.# x 50 µl E. coli MV1184 Electro-Cells Cat.# x 50 µl E. coli DH5a Electro-Cells* Cat.# x 50 µl E. coli HST02 Electro-Cells Cat.# x 50 µl Application Used for Transformation of DNA into E. coli by electoroporation Electroporation is the application of a brief, high voltage electric pulse to the E. coli cells, leading to the formation of pores in the plasma membrane. This allows DNA to be taken directly into the cell cytoplasm through these pores. Transformation of E. coli by electoroporation is a reliable method to obtain high transformation efficiencies of competent cells. The efficiencies are generally higher than those achieved by transformation via calcium ion based cell techniques. This technique reduces the time required for experiments, and is especially useful in introducing a small sample amount into cells. Takara offers five strains of Electro-Cells prepared with our original high transformation efficiency method. (E. coli HB101, JM109, MV1184, DH5α) *E.coli DH5α competent cells are not available in the U.S. Cell Density > 1 x bacteria/ml Purity When inoculating 100 µl of E. coli competent cells on LB-agar medium containing ampicillin at 100 µg/ml, colonies are not formed 80 C References 1. Hanahan, D. (1983) J. Mol. Biol., 166: Messing, J. (1985) Gene, 33: Kunkel, T. A., et al. (1987) Methods in Enzymology 154: Kramer, W. and Frits, H. J. (1987) Methods in Enzymology 154: Hashimoto-Gotoh, T., et al. (1993) Gene 137: Dower, W. J., Miller, J. F. and Ragsdale, C. W. (1988) Nucl. Acids Res., 16: Bottger, E. C. (1988) BioTechniques 6: 878. Purity When inoculating 50 µl of E. coli electro-cells on LB-agar medium containing ampicillin at 100 µg/ml, colonies are not formed 80 C References 1. Hanahan, D. (1983) J. Mol. Biol. 166: Messing, J. (1985) Gene 33: Kunkel, T. A., et al. (1987) Methods in Enzymology 154: Kramer, W. and Frits, H. J. (1987) Methods in Enzymology 154: Hashimoto-Gotoh, T., et al. (1993) Gene 137: Dower, W. J., Miller, J. F. and Ragsdale, C. W. (1988) Nucl. Acids Res. 16: Bottger, E. C. (1988) BioTechniques 6:

58 Genotype E. coli HB101: supe 44, r(mcrc-mrr ), reca 13, ara -14, proa2, lacy1, galk2, rpsl20, xyl-5, mtl-1, leub6, thi-1 E. coli JM109: reca1, enda1, gyra96, thi-1, hsdr17(rk - mk + ), e14 (mcra ), supe44, rela1, r(lacproab)/f [trad36, proab +, lac I q, lacz r M15] E. coli CJ236: dut 1, ung 1, thi-1, rela1/pcj105(f 1 cam r ) E. coli BMH71-18 muts: r( lac-proab ), supe, thi-1, muts 215:: Tn10 (tet r )/F 1 [trad36, proab +, lac I q, lacz rm15] E. coli MV1184: ara, r(lac-proab), rpsl, thi (ø80lacz rm15), r(srl-reca)306:: Tn10 (tet r )/F [trad36, proab +, lac I q, lacz rm15] E. coli TH2: supe44, hsds20(rb mb ), reca13, ara-14, proa2, lacy1, galk2, rpsl20, xyl-5, mtl-1, thi-1, trpr624 E. coli DH5α: F -, ø80d lacz rm15, r(laczya-argf)u169, deor, reca1, enda1, hsdr17(rk, mk + ), phoa, supe44, l, thi -1, gyra 96, rela 1 E. coli HST02: F [trad36, pro A + B +, lac I q, lac Z r M15]/ r (lac-pro AB), rec A, end A, gyr A96, thi, e14 (mcr A ), sup E44, rel A, rdeo R, r (mrr-hsd RMS-mcr BC) E. coli HST04 dam-/dcm-: F --, ara, Δ(lac-proAB)[ø80dlacZΔM15], rpsl(str), thi, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, dam, dcm Bacterial Strains E. coli HB101: HB101 has a stable genetic phenotype and is used generally in recombinant DNA experiments. E. coli JM109: JM109 is used to select recombinant DNA using β-galactosidase activity (α-complementation) recovered with lacz α peptide of the vector DNA and lacz r M15 coded in the F episome in this strain. This strain carries F episome, and it is useful for preparation of ssdna as well as gene library construction and subcloning. E. coli CJ236: The host for preparation of a uracil-containing ssdna (Kunkel method). CJ236 contains dut and ung, and is deficient in the enzymes, dutpase (Dut) and Uracil-DNA-glycosylase (Ung). These deficiencies lead to misincorporation of deoxyuridine in place of some thymidine residues. Kunkel s site-directed mutagenesis method utilizes this strain as the host of puc118/119 or M13 phage to prepare template ssdna. A F episome (pcj105) is maintained stably in the presence of chloramphenicol because of chloramphenicol resistance gene. E. coli BMH71-18 muts: Host for site-directed mutagenesis. BMH71-18 muts contains muts and is deficient in the ability to repair the mismatched DNA. Therefore, this strain is used in site-directed mutagenesis to suppress the repair of mismatched sites and increase mutagenesis efficiency. E. coli MV1184: Host for the selection of amber mutations. MV1184 is an amber suppressor free strain. Therefore, this strain is used for the selection of ambermutated DNA. When using Mutan -Express Km (6090/6091) or Mutan -Super Express Km (RR022) for site-directed mutagenesis, this strain is used for the selection of mutated DNA. Additionally this strain contains a F 1 episome and can be used to prepare ssdna as a host for M13 phage vector or phagemid vector. E. coli TH2: Host for Enforcement cloning system pkf3s (6086). TH2 has the mutations in supe, trpr and rpsl, and is suitable for the selection of foreign genes in a medium containing streptomycin, using the pkf3 vector which contains the lethal gene rpsl at the downstream of trp promoter. E. coli DH5α: Host for Blue/White screening utilizing the β-galactosidase activity (a-complementation) in combination with puc vectors. As this strain does not carry lac l q, IPTG is not needed. Therefore, DH5α allows easy selection of recombinant DNA with X-Gal when constructing gene libraries or subcloning recombinant plasmid. E. coli HST02: Host for construction of libraries, subcloning. HTS02 completely lacks in (mrr-hsd RMS-mcr BC) and mcr A. Therefore, it can widely be used in construction of gene libraries and subcloning of methylated DNA. It can be used in blue/white selection by a-complementation. It can also be used as host for M13 vector DNA since it contains a F plasmid. E. coli HST04 dam-/dcm-: E. coli HST04 dam-/dcm- lacks the genetic factors dam and dcm found in wild type E. coli that are necessary for DNA methylation. Plasmids prepared using this product can be cut by restriction enzymes which are normally blocked by dam or dcm methylation. A dam/reca double mutation is lethal, so this strain is reca +.Therefore, transformation of extracellular DNA with repeat sequences can result in recombination by reca. Therefore, this product is not suitable for routine DNA cloning other than preparation of unmethylated plasmids as mentioned above. Product Specifications 1. Transformation Efficiency (Competent Cells): When using 1 ng plasmid DNA for transformation: 100 µl HB101 Competent Cells >1 x 10 8 cfu/µg pbr µl JM109 Competent Cells >1 x 10 8 cfu/µg pbr µl CJ236 Competent Cells >1 x 10 7 cfu/µg puc µl BMH71-18 muts Competent Cells >1 x 10 7 cfu/µg puc µl MV1184 Competent Cells >1 x 10 7 cfu/µg puc µl TH2 Competent Cells >1 x 10 7 cfu/µg pbr µl DH5a Competent Cells >1 x 10 8 cfu/µg puc µl HST02 Competent Cells > 1 x 10 8 cfu/ug puc µl HST04 dam-/dcm- Competent Cells > 1 x 10 6 cfu/ug puc19 2. Transformation Efficiency (Electro-Cells): When using 10 pg plasmid DNA for electroporation: 50 µl HB101 Electro-Cells >5 x 10 8 cfu/µg pbr µl JM109 Electro-Cells >1 x 10 9 cfu/µg puc19 50 µl MV1184 Electro-Cells >1 x 10 8 cfu/µg puc µl DH5a Electro-Cells >1 x 10 9 cfu/µg puc19 50 µl HST02 Electro-Cells > 1 x 10 9 cfu/ug puc19 3. Stability of F plasmid, confirmation of a-complementation: E. coli JM109, E. coli BMH71-18 muts, E. coli MV1184, E. coli HST02. Less than 1% of white colonies appeared when transforming with a puc19 plasmid vector (with JM109) or puc119 (with BMH71-18 muts or MV1184) followed by plating on L-agar medium containing 100 µg/ml ampicillin, 0.2 mm IPTG, and 40 µg/ml X-Gal. E. coli CJ236 Strains lacking the F plasmid (pcj105) will not grow on a plate containing chloramphenicol at 30 µg/ml. E. coli DH5α Blue colonies appeared when puc19 DNA was transformed, and the transformants plated are on a L-agar medium containing 100 µg/ml of ampicillin and 40 µg/ml X-Gal. MOLECULAR BIOLOGY 2 Cloning, Mapping & cdna Synthesis 57

59 G418-resistant colony (count) G418-resistant colony (%) G418-resistant colony (%) 2MOLECULAR BIOLOGY Gene Transfer and Expression RetroNectin Reagent and RetroNectin Precoated Dishes Recombinant Human Fibronectin Fragment CH-296 RetroNectin Recombinant Human Fibronectin Fragment Cat.# T100A 0.5 mg protein (0.5 ml) RetroNectin Recombinant Human Fibronectin Fragment Cat.# T100B 2.5 mg protein (2.5 ml) RetroNectin Pre-coated Dish Cat.# T110A 10 precoated dishes Feature Enhanced efficiency: of Retroviral or Lentiviral-mediated gene transfer into suspended and adherent mammalian cells Improved success: in hard-to-infect cell types and stem cells Nontoxic: does not require cytotoxic polybrene Superior performance: provides superior transduction efficiency compared to fibronectin, polybrene or protamine Use less virus: RBV method results in greater efficiency enhancement and enables use of small volumes of virus solution RetroNectin (CH-296) is a chimeric peptide of recombinant human fibronectin fragments produced in E. coli, consisting of three functional domains: a central cellbinding domain (type III repeat, 8 10), heparin-binding domain II (type III repeat, 12 14), and a CS1 site within the alternatively-spliced IIICS region. It is a 574 amino acid protein with a molecular weight of 63 kda. Fibronectins (FNS) are multifunctional cell adhesive glycoproteins present in the extracellular matrix and plasma. When coated on the surface of containers such as culture dishes, petri dishes, flasks or bags, RetroNectin significantly enhances retrovirus-mediated gene transduction into mammalian cells. This enhancement is hypothetically due to co-localization of retroviral particles and target cells on the molecules of RetroNectin. Virus particles bind RetroNectin via interaction with heparin-binding domain II, and target cells bind mainly through the interaction of the cell surface integrin receptor VLA-4 with the fibronectin CS1 site. Additionally, cells may also bind through the interaction of another fibronectin ligand (RGDS in repeat 10) within the central cell-binding domain with a corresponding integrin receptor VLA-5 on the cell surface. : Cat.# T100A/B: 20 C, Cat.# T110A: 4 C Form: Sterilized Solution in 12.5 mm sodium citrate (ph6.2) and 1.25% sucrose Concentration: 1 µg/µl Source: E. coli expressing human fibronectin fragment CH-296 Molecular weight: 62,613 (amino acid sequence) Purity: >90% by HPLC Note Repeated freeze-thaw cycles are limited less than 10 times. Do not mix vigorously (Do not use vortex mixer). Plate coating RetroNectin is coated onto a plate at a concentration of µg/ml to cover a plate at 4 to 20 µg/cm 2. When 2 ml of RetroNectin solution at a concentration of µg/ ml is plated into 3.5 cm dish, one vial (containing 0.5 mg) is sufficient to coat 2 to 12 dishes (each 3.5 cm in diameter, 10 cm 2 area). Note Do not filter the diluted solution ( µg/ml). The diluted solution ( µg/ml) should not be stored. Cell Efficiency of Gene Transfer (%) TF HEL 28.1 NIH/3Y CD34+ bmc 86.9 Mice BM MNC fraction 29.2 K HL Examples of Cell Transduction Efficiency with RetroNectin RetroNectin RetroNectin BM BSA BSA Retroviral Transduction of neo r Gene into c-kit Positive Murine Bone Marrow Cell using RetroNectin (in vitro assay). RetroNectin CB BSA Retroviral Transduction of neo r Gene into CD-34 Positive Cells using RetroNectin BM=CD-34-enriched bone marrow; CB=umbilical cord blood cells. 0 RetroNectin BSA Retroviral Transduction of neo r Gene into an Established Cell Line (NIH/3T3) Cells using RetroNectin. 58

60 Hypothesized Mechanism of Action of RetroNectin Target Cell MOLECULAR BIOLOGY VLA-5 (α 5 β 1 ) receptor Retrovirus vector VLA-4 (α 4 β 1 ) Cell binding domain (C-Domain; RGDS) Heparin binding domain (H-Domain) Structure of Fibronectin and RetroNectin CS-1 2 NH2 Fibronectin Fibrin Heparin Collagen (DNA) Cell Heparin Cell Fibrin (IIICS) RetroNectin (CH-296) SH RGDS VLA5 C CS-1 Retrovirus vector VLA4 H CS-1 SH SS COOH Gene Transfer and Expression Fibronectins (FN) are Multifunctional Cell Adhesive Glycoproteins Present in Extracellular Matrix and Plasma. RetroNectin is a 574 aa (63 kda) chimeric peptide of recombinant human FN fragments. It includes the central cell-binding domain (type III repeat, 8, 9, 10, binds VLA 5), heparin-binding domain II (type III repeat, 12, 13, 14, binds retrovirus), and the CS-1 site (binds VLA 4) of fibronectin. (%) RetroNectin Polybrene Protamine Using RetroNectin with Human Stem Cells The graph shows the viral transduction efficiency (measured by expression of the recombinant fluorescent protein) obtained using the RetroNectin method, the Polybrene method, and the Protamine method. These results demonstrate that RetroNectin enables and enhances retrovirus-mediated gene transfer into stem cells that express lower levels of GALV envelope receptor, such as hcd34+ or hadsc. 0 hcd34 + hmsc hadsc Comparison of Retrovirus-Mediated Gene Transduction Efficiency in Various Methods into Human Stem Cells. 59

61 2MOLECULAR BIOLOGY Gene Transfer and Expression pmei-5 DNA pmei-5 Neo DNA Cat.# µg pmei-5 DNA Cat.# µg Retrovirus-Mediated Gene Transfer into Mammalian Cells This product is a plasmid vector for producing a high expression efficiency retrovirus vector. By transfecting this vector into an appropriate packaging cell line, the vector transiently or stably expresses transcribed products containing the virus-packaging signal (Ψ), target gene and selective marker. This vector possesses LTR and Ψ (viral packaging signal), but not the structural genes necessary for particle formation and replication (sequences gag, pol, and env). Because this vector contains an intron with high splicing capacity, it yields high transcription efficiency. pmei-5 Neo DNA (Cat.# 3655) contains the neomycin pbapo-cmv Vector series pbapo-cmv is a simple gene expression vector for mammalian cells. This vector has a promoter from cytomegalovirus (CMV IE promoter) and polya signal from herpes simplex virus thymidine kinase. The expression plasmid for the target gene can be constructed by inserting the target gene ORF into the cloning site. This vector can also be used to express mirna precursors and other transcription products in addition to protein-coding transcripts. Furthermore, the cassette of [promoter + ORF + polya signal] can be removed from this vector and easily transferred to an adenovirus vector. Having a high infection efficiency and a wide target cell spectrum, adenovirus vectors are suitable for in vitro and in vivo gene transduction. For constructing recombinant adenoviruses, the Adenovirus Dual Expression Kit (Cat. # 6170) is recommended. resistance gene as a selective marker. Gene expression two to eight-fold higher than pdon-al-2 series can be expected. Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Vector Sizes pmei-5 Neo DNA: 5,820 bp pmei-5 DNA: 4,689 bp 20 C pbapo-cmv Neo DNA Cat.# µg pbapo-cmv Pur DNA Cat.# µg pbapo-cmv DNA Cat.# µg In addition to a basic vector, the pbapo-cmv series also includes vectors that bear a neomycin resistance gene (Cat. #3240) or a puromycin resistance gene (Cat. # 3241) for stable expression in mammalian cells. Components 3240 and 3242 pbapo-cmv DNA (Cat.# 3242) pbapo-cmv Neo DNA (Cat.# 3240) pbapo-cmv Pur DNA (Cat.# 3241) 20 C 20 µg (Concentration: 0.5 µg/µl) 20 µg (Concentration: 0.5 µg/µl) 20 µg (Concentration: 0.5 µg/µl) 60

62 pdon-ai-2 DNA pdon-ai-2 Neo DNA Cat.# µg pdon-ai-2 DNA Cat.# µg Retrovirus-Mediated Gene Transfer into Mammalian Cells pdon-ai-2 DNA enables production of a retrovirus vector with high transfer efficiency. By transfecting the vector into an appropriate packaging cell line, the vector expresses transient or stable transcribed product containing virus-packaging signal (Ψ) and target gene. This vector possesses LTR and Ψ (viral packaging signal), but not the structural gene necessary for particle formation and replication, which are gag, pol, and env, and contains HCMV IE promoter within the U3 region of 5 LTR. pdon-ai-2 Neo DNA contains neomycin resistant gene as a selective marker. The production of 2-8-fold higher-titer virus can be achieved by transfecting a version of pdon-ai-2 DNA containing a target gene compared using conventional retrovirus vector. This product is a modified plasmid of pdon-ai DNA which was developed by Dr. S. Kim 1). 20 C Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Chain length pdon-ai-2 Neo DNA : 5,719 bp pdon-ai-2 DNA : 4,586 bp Preparation cccdna (covalently closed circular DNA) was purified by CsCl-EtBr ultracentrifugation. Purity Confirmed to maintain cloning sites by dideoxy sequencing method. Confirmed to be cleaved at a single site by restriction enzymes PmaC I, Apa I, Sac II, Not I, Bgl II, Cla I, BamH I, Sal I and Hpa I. Reference 1. Kim, H. S., Yu, S. S., Park, S. J., Robbins, D. P., An, S. C. and Kim, S. (1998) Journal of Virology, 72, MOLECULAR BIOLOGY 2 pdon-5 DNA pdon-5 Neo DNA Cat.# µg pdon-5 DNA Cat.# µg Retrovirus-Mediated Gene Transfer into Mammalian Cells This product is a plasmid vector for producing a retrovirus vector with high transfection, expression efficiency. By transfecting the vector into an appropriate packaging cell line, the vector expresses transient or stable transcribed product containing viruspackaging signal (ψ) and target gene and selective marker. This vector possesses LTR and ψ (viral packaging signal), but not the structural gene necessary for particle formation and replication, which are gag, pol, and env, and this vector contains HCMV IE promoter within U3 region of 5' LTR and neomycin resistant gene as a selective marker. HCMV IE promoter has high promoter activity, and can produce high-titer retroviruses. Also this vector contains intron with high ability of splicing, and it yields high transcription efficiency. 20 C Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Chain length pdon-5 Neo DNA : 5,828bp pdon-5 DNA : 4,697 bp Preparation cccdna (covalently closed circular DNA) was purified by CsCl-EtBr ultracentrifugation. Purity Confirmed to maintain cloning sites by dideoxy sequencing method. Confirmed to be cleaved at a single site by restriction enzymes PmaC I, Apa I, Sac II, Not I, Bgl II, Cla I, BamH I, Sal I, Hpa I and Xho I. Gene Transfer and Expression 61

63 2MOLECULAR BIOLOGY Gene Transfer and Expression pbapo-ef1 a Vector series pbapo-ef1 α Neo DNA Cat.# µg pbapo-ef1 α Pur DNA Cat.# µg pbapo-ef1a is a simple gene expression vector for mammalian cells. This vector carries a promoter from human polypeptide chain elongation factor (EF-1a promoter) and a polya signal site from herpes simplex virus thymidine kinase gene. The vectors are useful for the construction of expression plasmids by inserting the ORF of a target gene at the multicloning site. This vector can also be used to express mirna precursors and other transcripts in addition to ordinary genes. Furthermore, a cassette of [promoter + ORF + polya signal] can be easily removed from this vector and transferred to an adenovirus vector. Adenovirus vectors, which have a high infection efficiency and a wide target cell spectrum, are suitable for in vitro and in vivo gene transductions. For constructing recombinant adenoviruses, the Adenovirus Dual Expression Kit (Cat.# 6170) is recommended. The pbapo-ef1a series includes vectors carrying a neomycin-resistance gene (Cat. #3243) or a puromycin resistance gene (Cat.# 3244) as a selection marker in mammalian cells. Takara s Adenovirus Expression Vector Kit (Dual Version) offers two different methods for recombinant adenovirus production using the same stable cosmid vector. The Full-Length DNA Transfer Method produces recombinant adenoviruses using recombinant cosmids that have been created by direct insertion of the target gene into the cosmid, i.e., no shuttle vector is required. Alternatively, with the COS-TPC method, an adenovirus DNA fragment associated with a terminal protein complex is co-transfected along with a cosmid carrying the target gene into HEK 293 cells. The COS-TPC Method may be performed in situations where, due to the characteristics of the gene, use of the Full-Length DNA Transfer Method may not result in desired recombinant virus production. The COS-TPC Method offers increased efficiency of recombinant adenovirus production (up to two orders of magnitude) as compared to conventional methods. The Adenovirus Expression Vector Kit (Dual Version) contains components for both Full-Length DNA Transfer and COS-TPC Methods (except DNA-TPC for the COS-TPC Method, which is sold separately). Components 3243 and 3244 pbapo-ef1α Neo DNA (Cat.# 3243) pbapo-ef1α Pur DNA (Cat.# 3244) 20 C 20 µg (Concentration: 0.5 µg/µl) 20 µg (Concentration: 0.5 µg/µl) Adenovirus Expression Vector Kit, Adenovirus Genome DNA- TPC and Adenovirus Dual Expression Kit (EF1a) Adenovirus Expression Vector Kit (Dual Version) Cat.# kit (5 reactions) Adenovirus Dual Expression Kit (EF1α) Cat.# reactions Adenovirus Genome DNA-TPC Cat.# µl (5 reactions) Kit Components 6170 and 6174 Adenovirus Expression Vector Kit (Dual Version) Cosmid Vector paxcwit 1 (0.3 µg/µl) 25 µl Cosmid Vector paxcawtit 2 (0.3 µg/µl) 25 µl Smi I (Swa I) (10 U/µL) 20 µl 10X H Buffer 100 µl BspT104 I (10 U/µL) 30 µl 10X L Buffer 100 µl DNA Dissolution Buffer 50 µl Ligation Solution 50 µl 10X TNE 2 1 ml Proteinase K (20 mg/ml) 200 µl 10% SDS 200 µl Control Cosmid paxcailaczit 3 (0.3 µg/µl) 50 µl 1 With no promoter 2 With CAG promoter 3 paxcawtit with β-gal gene insertion 6170 and 6174: 10% SDS: Thaw at 37 C and store at room temperature. Others: 20 C 6171: Adenovirus Genome DNA-TPC: 80 C (Note: The efficiency may be decreased by repeated freeze-thaws. Avoid uneccessary freeze-thaw cycles.) 62

64 Yeast Transformation System: Aureobasidin A Aureobasidin A Cat.# mg Aureobasidin A Cat.# 9000B 5 10 mg Aureobasidin A is a cyclic depsipeptide antibiotic with a molecular weight of approximately 1,100 Da. It is isolated from Aureobasidium pullulans R106. Aureobasidin A is toxic to filamentous fungi and yeast at low concentrations ( µg/µl), including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Aspergillus nidulans. Form The antibiotic is supplied lyophilized and should be reconstituted in ethanol or methanol at a stock concentration of mg/ml. It is nearly insoluble in water. Aureobasidin A Vectors paur Primers paur123 R Primer Cat.# pmol For DNA Sequencing and Cloning using paur DNA The paur Primers are used with paur DNA. Available as a sequencing primer for DNA inserts cloned into paur123 DNA (paur123 F/R Primer). Stock solutions should be stored at 4 C. References 1. Takesako, K. et al. (1993) J. Antibiot. 46: Nagiec, M.M. et al. (1995) J. Biol. Chem. 272: paur101 DNA Cat.# µg paur112 DNA Cat.# µg paur123 DNA Cat.# µg paur224 DNA Cat.# µg paur135 DNA Cat.# µg paur316 DNA Cat.# µg Cloning and Transformation in Yeast Systems Using Aureobasidin A as a Selective Antibiotic The paur vector series includes six E. coli-yeast shuttle vectors, each constructed for a particular application in either Saccharomyces cerevisiae, Schizosaccharomyces, Saccharomyces pombe or Aspergillus nidulans. The vectors include a novel drugresistance selective marker that confers Aureobasidin A resistance in transformed yeast. For more details on these vectors, please visit Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Concentration 3600, 3604: 1 µg/µl; 3605: 0.5 µg/µl Vector Size 3600: 6,687 bp 3603: 7,638 bp 3601: 7,104 bp 3604: 6,074 bp 3602: 6,982 bp 3605: 11,663 bp Form Lyophilized MOLECULAR BIOLOGY 2 Gene Transfer and Expression Pyrithiamine Vectors pptr I DNA Cat.# µg pptr II DNA Cat.# µg The pptr vectors are E. coli-aspergillus shuttle vectors which contain an ampicillin resistance gene (Amp R ) as the selection marker for E. coli and a pyrithiamineresistance gene (ptra) as the marker for Aspergillus. Since the multiple cloning site precedes the lacz region, any DNA insertion into this vector can be easily verified using plates containing IPTG and X-Gal. Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Vector Size 3621: 4,782 bp 3622: 10,032 bp Manufactured by Hakutsuru Sake Brewing Co., Ltd. 63

65 2MOLECULAR BIOLOGY RNA/Protein/DNA Extraction Dr. GenTLE Systems (Gene Trapping by Liquid Extraction) Dr. GenTLE System (Whole Blood) Cat.# reactions Dr. GenTLE Precipitation Carrier Cat.# µl (200 tests) Dr. GenTLE High Recovery (from Yeast) Cat.# reactions Quick (40 minutes), Consistent Extraction of High-Purity DNA from Whole Blood Extraction of DNA from Whole Blood Pretreated with Anti-Coagulants Such as Citric Acid, EDTA and Heparin Extraction of DNA from Infectious Samples Takara s Dr. GenTLE Systems are designed to enable the extraction of highly purified DNA from whole blood (Cat.# 9081) or yeast (Cat.# 9082) for use in various reactions. The standard protocol describes extraction from 100 μl of whole blood, but the kit can also be used for larger-volume extraction. The protocol is suitable for simultaneously processing a large number of samples. Both the Dr. GenTLE whole blood or yeast kits are composed of three solutions. Dr. GenTLE Solution I lyses cells immediately after being added to the sample, and a noncharged complex is formed with nucleic acids. The complex is then collected by centrifugation, and the precipitate washed with Dr. GenTLE Solution II. Dr. GenTLE Solution III is added to isolate only DNA, which can be precipitated by the addition of isopropanol. The protocol takes just 40 minutes and yields highly purified DNA. Use of the Dr. GenTLE Precipitation Carrier in ethanol precipitation results in higher recoveries of dilute (5 ng/ml) DNA samples than glycogen. Also, low temperature incubation is not required, and the white pellet generated is easily visible, preventing loss during washing. Kit Components 9081 Dr. GenTLE Solution I 2 50 ml Dr. GenTLE Solution II 4 50 ml Dr. GenTLE Solution III 2 50 ml 9094 Dr. GenTLE Precipitation Carrier µl 3M CH 3 COONa 2 1 ml 9082 Dr. GenTLE Yeast Solution A 15 ml Dr. GenTLE Yeast Solution B 3 ml Dr. GenTLE Yeast Solution C 6 ml TE Buffer 6 ml Notes 1. Dr. GenTLE System enables extraction of DNA from blood samples stored at 80 C. If the sample has been repeatedly freeze-thawed, lower DNA yields will occur. 2. The extraction of mitochondrial DNA has been confirmed using primers specific for mtdna. 3. The kits have not been tested for use with samples from HIV infected subjects. 9081: Room Temperature Dr. GenTLE Solution I may precipitate when stored below 20 C. If that occurs, heat to C for 1 2 hours to dissolve completely before use. Do not expose to temperatures above 60 C. Incubation >60 C may result in deterioration of Dr. GenTLE Solution I. 9094: Room Temperature 9082: 4 C Only Dr. GenTLE Yeast Solution B can be stored at room temperature. When stored at low temperature, precipitates may appear. If precipitates appear, dissolve precipitates completely by heating at 37 C before use. 64

66 Protein and RNA Extraction Kit for Mammalian Cells (PAREx Kit) Protein and RNA Extraction Kit for Mammalian Cells (PAREx Kit) Cat.# MK reactions Simultaneous Extraction of Total RNA and Protein from Cultured Mammalian Cells Allows Screening of RNAi Knockout Experiments Both RNA and Protein Expression Features Simple and Quick Protocol: a total RNA and protein solution can be prepared in ~15 minutes; a total RNA-only solution can be obtained with an additional isopropanol precipitation step in 50 minutes Simultaneous Extraction of Total RNA and Protein from the Same Cellular Material: useful for RNAi effect analysis and RNA/protein expression analysis No Solvents Used (e.g. phenol/chloroform) PAREx (Protein and RNA Extraction Kit) allows quick, simultaneous extraction of both total RNA and protein from cultured mammalian cells without the use of phenol or chloroform. Simultaneous extraction of total RNA and protein from the same sample simplifies protocol steps and is desirable for increased consistency in RNAi knockout analyses and RNA and protein expression analysis applications. PAREx uses a cell lysis buffer containing a nonionic surfactant for solubilizing cells and removes DNA and residual cells through a salting-out method. Preparation of the solubilized cell solution containing total RNA and protein, obtained from cells, can be accomplished in ~15 minutes. This cell solution can be used directly for electrophoretic migration and Western blot analysis of proteins. By further addition of isopropanol to the solution, RNA can be removed and then resolubilized in an RNase-free solution. This RNA solution is suitable for use in realtime RT-PCR reactions. Kit Components MK700 Cell Lysis Buffer Denature Solution RNA Preparation Water 2 8 C 1 25 ml 3 25 ml 1 25 ml MOLECULAR BIOLOGY 2 Yeast Processing Reagent (for total RNA preparation) Yeast Processing Reagent (for total RNA preparation) Cat.# reactions Features Excellent for RNA Extraction in Yeast Enzymatic Gentle Yeast Cell Wall Disruption: without glass beads or liquid nitrogen Takara s Yeast Processing Reagent (for total RNA preparation) is a pretreatment reagent to enable isolation of high-purity total RNA from yeast. The pretreatment of yeast cells with this product involves: 1. washing and recovery of yeast cells by centrifugation; and 2. degradation of the yeast cell wall using the Yeast Processing Enzyme Solution (cell wall degradation enzyme) and Yeast Processing Buffer. Kit Components: 9089 Yeast Processing Buffer Yeast Processing Enzyme Solution RNase-free DNase I 10X DNase I Buffer 20 C 1.6 ml 160 μl 120 μl 80 μl RNA/Protein/DNA Extraction Miniprep DNA Purification Set Miniprep DNA Purification Set Cat.# reactions Kit Components: Quick and Simple Isolation of Plasmid DNA Cell Suspension Buffer 20 ml Cell Lysis Buffer 20 ml Neutralization Solution 30 ml The Miniprep DNA Purification Set is designed to allow simple, quick and reliable Wash Buffer* 20 ml isolation of plasmid DNA, utilizing a DNA binding silica membrane. This system is Miniprep Spin Column 50 pcs suitable for efficient isolation of plasmid DNA of less than 20 kb in size. The purified Collection Tube (2 ml) 50 pcs Alkaline Protease Solution 550 μl plasmid can be used directly for DNA sequencing as well as other molecular biology Nuclease-free dh techniques, such as enzyme reactions, without further manipulation. This kit 2 O 13 ml * Prior to use, prepare Wash Buffer up to 55 ml by adding 35 ml of ethanol. procedure generally yields 1 20 μg of plasmid DNA from 1 10 ml of E. coli overnight cultures. Because this kit utilizes alkaline protease, highly purified plasmid DNA without contaminating nuclease can be prepared from enda+ E. coli strains, i.e. Room Temperature HB

67 TaKaRa DEXPAT (DNA Extraction from Paraffin-embedded Tissue) 2MOLECULAR BIOLOGY TaKaRa DEXPAT Cat.# reactions Quick (25 minute), One-Step Extraction of PCR-ready DNA (< 400 bp) from Paraffin-Embedded Tissues Without Exposure to Hazardous Chemicals Extraction of DNA from Sections After Lengthy (<10 years) Efficient Extraction of DNA from Tissue Sections or Histology Slides This reagent is designed to enable one-step extraction of PCR-ready DNA from paraffin-embedded tissues fixed in 10% formalin. The one-step extraction protocol eliminates the laborious deparaffinization, organic solvent extraction and enzymatic digestion steps required in standard protocols. DEXPAT Reagent preparation of PCR-ready DNA takes only 25 minutes, versus 2 3 days for conventional extraction methods. Since DNA extracted from paraffin-embedded tissue may be somewhat degraded during formalin fixation or paraffin embedding, amplification of products greater than 400 bp in length is not recommended. The quality of the extracted DNA will be strongly dependent on the condition of the original embedded tissue. Kit Components 9091 DEXPAT Reagent 4 C (Shipped at room temperature.) References 1. Goelz, S.E. et al. (1985) BBRC, 130, No.1, Sato, Y. et al. (1990) Am. J. Pathol., 136, 267. Related Product TaKaRa Ex Taq (Cat.# RR001A) 250 units Easy Extraction from paraffin-embedded tissues 5 10 ml RNA/Protein/DNA Extraction TaKaRa DEXPAT Easy TaKaRa DEXPAT Easy Cat.# reactions DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues for use in PCR. Features Easy: no deparaffinizing step Fast: only 30 minutes for the entire process Pure Template: PCR template-grade DNA (for amplification of DNA <400 bp) No Toxic Substances Easy Recovery: good separation of the DNA aqueous solution and the absorbent resin allows for easy recovery of DNA aqueous solution TaKaRa DEXPAT Easy is designed for simple and rapid extraction of PCR-ready DNA from Formalin Fixed Paraffin-Embedded (FFPE) tissues. This kit provides a readyto-use optimized mixture of a special surfactant and PCR inhibitor-absorbent resin predispensed in 1.5-mL microtubes. Simply add paraffin-embedded tissue sample into one of the microtubes, incubate at 100, centrifuge at 4 and cool on ice for 5 minutes to obtain PCR-ready DNA in approximately 30 minutes. The refrigerated centrifugation following heat treatment allows the formation of a gel-like layer above the absorbent resin. When collecting the DNA solution, the gel-like layer prevents contamination of the absorbent resin into the DNA solution. The absorbent resin may inhibit PCR amplification. Extraction of DNA from paraffin-embedded tissue sections using conventional methodology is a time-consuming and labor intensive process. TaKaRa DEXPAT Easy, however, offers a rapid and simplified procedure. This reagent retains the performance of TaKaRa DEXPAT (Cat.# 9091), and has the added benefits of being predispensed in reaction tubes and has an improved composition designed to separate the DNA aqueous solution and the absorbent resin during DNA recovery. Kit Components 9104 TaKaRa DEXPAT Easy 4 C 500 µl 10 microtubes 5 bags 66

68 TaKaRa RECOCHIP TaKaRa RECOCHIP Cat.# pieces Recovery of DNA from Agarose Gels The RECOCHIP is a disposable chip designed to recover DNA fragments (up to 23 kb) from agarose gels. It consists of a non-woven polyester backing attached to a cellulose dialysis membrane. After visualization of the band of interest, the RECOCHIP is inserted directly into the gel ahead of the band. The band is then run into the space created between the backing and the dialysis membrane. DNA is recovered by centrifuging the chip in a 2.0 ml tube. For size specifications and electrophoresis recommendations, see our web site at Nominal Fractionating Size 12,000 14,000 Da (=18-21 base pairs or bases) Room temperature Kit Components 9039 RECOCHIP 100 pieces 2.0 ml tube 100 pieces Gel Cutter 3 pieces Manufactured by Toyo Roshi Kaisha, Ltd. Black surface Target DNA band Cathode (-) RECOCHIP Gel tray Agarose gel Anode (+) Diagram showing position of RECOCHIP in electrophoresis apparatus. MOLECULAR BIOLOGY 2 SUPREC -01 DNA Purification Cartridge SUPREC -01 DNA Purification Cartridge Cat.# cartridges Rapid Recovery of DNA from Agarose Gels SUPREC -01 filter cartridges are designed for rapid recovery of DNA from agarose gels. After the DNA is separated by gel elec tro phore sis, the relevant gel slice is excised and in sert ed into the cartridge. The car tridge is then centrifuged. The DNA will elute out of the gel and pass through the car tridge filter into the lower portion of the tube. One car tridge can process up to 0.4 cm 3 of agarose gel and isolate microgram quantities of DNA. The DNA will be re cov ered efficiently and is suitable for subsequent manipulations such as cloning, sequencing or enzymatic cleavage and modifications. Room temperature Spin DNA agarose gel slice filter Agarose filtrate DNA solution RNA/Protein/DNA Extraction 67

69 2MOLECULAR BIOLOGY Plant Products Fruit-mate for RNA Purification Fruit-mate for RNA Purification Cat.# ml Removal of Polysaccharides or Polyphenols from Prepared Plant RNA Features Pre-treatment reagent for extracting total RNA from plants with: high polysaccharide or polyphenol content Successful results confirmed with: strawberry, cherry tomato (fruit), banana (fruit), tomato (seed), rice (seed), potato (rhizome), mandarin orange (peel), pine (leaf), aloe (leaf), mango (fruit), and Arabidopsis (seed) Fruit-mate for RNA Purification is used when extracting total RNA from plant samples (root crop, fruit, seed, etc.) that contain large quantities of polysaccharides or polyphenols. Fruit-mate contains a non-ionic polymer that binds to substances such as polysaccharides and polyphenols. Once bound these substances can then be easily removed by centrifugation. RNA extraction from high polysaccharide or highpolyphenol sample, which are normally difficult to use for RNA extraction using standard methods, can be performed effectively with Fruit-mate. Room temperature High-Salt Solution for Precipitation (Plant) Excellent for DNA extraction from plants High-Salt Solution for Precipitation (Plant) Cat.# ml Removal of Residual Polysaccharides from RNA Solutions High-Salt Solution for Precipitation (Plant) is a reagent used to remove polysaccharide remaining in the RNA solution after an RNA preparation. It is possible to prepare high purity total RNA by simply adding this reagent during an isopropanol precipitation step. High purity sample is easily prepared. Note: Do not use for recovery of small RNA molecules. For small amounts of RNA, use Dr. GenTLE Precipitation Carrier (Cat.# 9094) during the isopropanol precipitation for increasing RNA recovery. Room temperature Form 1.2 M NaCl, 0.8 M Sodium Citrate Prepare high purity RNA from plants with this post-extraction treatment Plant DNA Isolation Kit Plant DNA Isolation Kit Cat.# reactions Features Simple Extraction of Genomic DNA No Mortar and Pestle needed to grind Plant Sample! Ready to Use Kit Isolation Complete in 30 Min. Takara Plant DNA Isolation Reagent is a ready-to-use kit that uses benzyl chloride for extracting plant genomic DNA. Benzyl chloride causes disintegration of plant cell walls by benzylation of the hydroxyl groups in cellulose and other components of the cell wall. This kit takes advantage of this property in a procedure that involves freeze thawing and physical grinding of plant tissue with a pipette tip several times in a microcentrifuge tube to achieve degradation of the cell wall. Traditional methods of plant cell wall disruption, which rely on pulverization in liquid nitrogen using a mortar and pestle, are not needed. This makes preparing multiple samples at the same time more convenient. Furthermore, improvements in reagent composition have reduced the heat treatment step to only 15 minutes, and the entire process through aqueous phase collection can be completed within 30 minutes. The genomic DNA obtained can be used directly in PCR reactions, or for restriction endonuclease digestion. Kit Components 9194 Extraction Solution 1 40 ml Extraction Solution 2 8 ml Extraction Solution 3 (100% Benzyl Chloride) 15 ml Room Temperature. Extraction Solution 2: a precipitate may form when stored at low temperature. The precipitate may be dissolved by heating to 50 C. Store at room temperature after dissolving precipitate. 68

70 Agrobacterium tumefaciens LBA4404 Electro-Cells Agrobacterium tumefaciens LBA4404 Electro-Cells Cat.# μl 5 tubes Features High-Quality Electrocompetent Cells: save time when performing your plant transformation experiments Facilitates Delivery of a Binary Vector Validated Plasmid Transformation Efficiency Agrobacterium tumefaciens (Rhizobium radiobactor) can transfer T-DNA (transfer DNA), which is part of its own Ti plasmid, into host plant cells and insert this DNA into the plant chromosomal DNA. The inserted genes on T-DNA are expressed. By utilizing this gene-transfer mechanism, the binary vector method was invented for plant transformation 1. In this system, the pathogenic genes of T-DNA in Ti plasmid are replaced with selective marker genes and the exogenous target gene, to transfer the target gene into plant chromosomal DNA by means of Agrobacterium -mediated gene transfer. Agrobacterium tumefaciens strain LBA4404 and the binary vector method were developed by Dr. P.J. Hooykaas at Leiden University in the Netherlands. This strain of Agrobacterium tumefaciens includes the pal4404 plasmid, which only contains the T-DNA vir region (genes responsible for vir gene induction and T-DNA transfer). LBA4404 is a widely used strain for plant transformation. This product contains competent cells for transformation by electroporation 3. Using A. tumefaciens and the binary vector method, you can transform various plants for in fection (transfection) experiments. Quality. The transformation efficiency was tested with 1 ng of pri900 DNA, and the colonies were selected in plates which included kanamycin and strepto mycin. Efficiency of > colonies/µg pri 900 DNA was obtained. See Section IV. Protocol in the User Manual for more information. Kit Components: 9115 Agrobacterium tumefaciens LBA4404 Electro-Cells 5 tubes 40 μl pri900 DNA a (1 ng/μl) 10 μl SOC media b 10 tubes 1 ml a pri900 DNA : pri 910 DNA (Cat.# 3261) without a multicloning site. b SOC media: 2% Tryptone; 0.5% Yeast extract; 10 mm NaCl; 2.5 mm KCl; 10 mm MgSO 4 10 mm MgCl 4 20 mm Glucose 80 C Reference 1. A. Hoekema, P. R. Hirsch, P. J. J. Hooykaas., and R. A. Schilperoort., (1983) Nature, 303, G. Ooms, P.J. J. Hooykaas, R. J. M. V. Veen, P. V. Beelen, T. J. G. Regensburg-Tuink, R. A. Schilperoort., (1982) Plasmid, 7, S. Wen-jun and B. G. Forde (1989) Nucleic Acids Research, 17 (20), 8385 MOLECULAR BIOLOGY 2 Plant Products 69

71 pri 909/910 DNA 2MOLECULAR BIOLOGY pri 909 DNA Cat.# µg pri 910 DNA Cat.# µg Agrobacterium-Mediated Plant Transformation (Binary Method) pri 909/910 DNA are binary vectors that have a region of T-DNA for plant transformation. This originated from Ri plasmid of Rhizobium (Agrobacterium tumefaciens) rhizogenes, lacking a vir region. pri 909/910 DNA are also shuttle vectors and replicate autonomously in E. coli and Rhizobium (Agrobacterium). In E. coli, these vectors are propagated at high copy number due to the presence of ColE1 ori. pr1909/910 are maintained stably in Rhizobium (Agrobacterium) due to mutant type replication origin of Ri plasmid (Ri-ori). These vectors include NPTIII to confer Kanamycin resistance as the selection marker for E. coli and Rhizobium (Agrobacterium), a mutant type kanamycin resistant gene NPTII as the selection marker for plant, and the same multi-cloning sites as puc type plasmids. The vectors are available for Agrobacterium-mediated binary plant transformation. Stable chromosomal integration of a target gene is possible because the cloning site is located at the position closer to Right Border(RB) of T-DNA than the selection marker (NPT II) for the plant, so the target gene is not deleted. Form Tris-HCl, ph 8.0 (10 mm); EDTA (1 mm) Preparation Purification by column treatment Base Pairs 9168 bp Purity Contains over 70% double-stranded cavalently closed circular form I (RF1). Shown to retain cloning sites by dideoxy sequencing method. Shown to be cleavable at a single site by restriction enzymes specific to these sites. 20 C pri 101 DNA Vectors Plant Products pri 101 ON DNA Cat.# µg pri 101 AN DNA Cat.# µg Features Easy, Stable Integration of Dicot and Monocot Target Genes into Plant Chromosomes Increased Expression of Target Gene by Translation Enhancer Region (5 -UTR derived from ADH) Increased Versatility: pri 101 AN is for dicotyledonous plant cells, pri 101 ON is for monoctyledonous plant cells pri 101 DNA series are binary vectors for expressing foreign genes in plant cells, which include the 35S promoter of cauliflower mosaic virus (CaMV) and a 5 noncoding region (5 -UTR) of alcohol dehydrogenase (ADH) gene. The 5 -UTR of ADH functions as a translational enhancer in plants. There are two types of vectors, pri 101-AN DNA having a 5 -UTR of Arabidopsis ADH (AtADH 5 -UTR) and pri 101 ON DNA having a 5 -UTR of rice ADH (OsADH 5 -UTR). The pri 101-AN is for dicotyledonous plants such as tobacco or Arabidopsis and the pri 101-ON is for monocotyledonous plants such as rice. The pri 101 DNA are shuttle vectors, and replicate autonomously in E. coli and Rhizobium (Agrobacterium). pri 201 DNA Series pri 201-AN DNA Cat.# µg pri 201-ON DNA Cat.# µg Binary Vectors for Plant Transformation Use pri 201-AN DNA to transform dicotyledonous plants. Use pri 201-ON DNA to transform monocotyledonous plants. The presence of 2 multicloning sites enable multigene transformation with a single vector. Vectors contain a replication origin (ColE1 ori) derived from puc plasmids, which allows maintenance at a high-copynumber in E. coli. pri 201 DNA series are binary vectors designed for expressing target genes in transformed plant cells. This series of vectors retain the backbone of pri 101 vectors (Cat.# 3262/3263), which includes an alcohol dehydrogenase (ADH) gene-derived 5 untranslated region (5 -UTR) (translational enhancer region) downstream of the 35S promoter from cauliflower mosaic virus (CaMV). In addition, they have a heat shock protein (HSP) gene-derived terminator in place of the nopaline synthase (NOS) gene-derived terminator, allowing higher target gene expression compared with the pri 101 series 2. Further, multigene transformation with a single vector is possible by integrating an expression cassette containing another gene (promoter + enhancer + gene of interest + terminator) into the cloning site (MCS2) downstream of the HSP terminator. The pri 201 series has two types of vectors: pri 201-AN DNA and pri 201-ON DNA. Carrying an Arabidopsis ADH-derived 5 -UTR (AtADH 5 -UTR), pri 201-AN DNA is suitable for dicotyledonous plants. With a rice ADH-derived 5 -UTR (OsADH 5 -UTR), pri 201-ON DNA is compatible with monocotyledonous plants. Components pri 201-AN DNA (Cat.# 3264) 10 µg pri 201-ON DNA (Cat.# 3265) 10 µg Shipping 20 C 70

72 Takara DNA Vectors and Genomic DNAs Takara DNA Vectors Product Catalog No. Quantity Notes Chain Length Concentration puc118 EcoR I/BAP µg (0.1 OD) 3,162 bp µg/ml puc118 BamH I/BAP µg (0.1 OD) 3,162 bp µg/ml puc118 Hinc II/BAP µg (0.1 OD) 3,162 bp µg/ml puc118 Pst I/BAP µg (0.1 OD) 3,162 bp µg/ml puc118 Hind III/BAP µg (0.1 OD) 3,162 bp µg/ml ptv118n DNA µg (0.5 OD) 3,163 bp µg/ml pkf 18k-2 DNA µg (0.2 OD) 2,204 bp µg/ml pkf 19k-2 DNA µg (0.2 OD) 2,204 bp µg/ml pstv28 DNA µg (0.5 OD) 2,999 bp µg/ml pstv29 DNA µg (0.5 OD) 2,999 bp µg/ml ptwv228 DNA µg (0.5 OD) 4,039 bp µg/ml Takara Genomic DNAs Product Catalog No. Quantity Notes Chain Length Concentration λ DNA µg (8 OD) λcl857 Sam7-source 48,502 bp µg/ml фx174 RF I DNA µg (0.5 OD) >70% RF I 5,386 bp µg/ml pbr322 DNA µg (0.5 OD) >70% RF I (2) 4,361 bp µg/ml M13 mp18 SS DNA (Virion DNA) µg (0.625 OD) >90% SS DNA 7,249 bp 0.2 µg/µl M13 mp18 RF DNA µg (0.2 OD) >70% RF I (1) 7,249 bp µg/ml puc 18 DNA µg (0.5 OD) (1) 2,686 bp µg/ml puc 19 DNA µg (0.5 OD) (1) 2,686 bp µg/ml phsg 298 (kan r puc vector) µg (0.5 OD) puc 18 MCS 2,675 bp µg/ml phsg 299 (kan r puc vector) µg (0.5 OD) puc 19-like MCS 2,673 bp µg/ml phsg 396 (Cm r puc vector) µg (0.5 OD) puc 19-like MCS (1,2) 2,238 bp µg/ml phsg 398 (Cm r puc vector) µg (0.5 OD) puc 18 MCS (1) 2,227 bp µg/ml puc 118 DNA µg (0.5 OD) (1) 3,162 bp µg/ml puc 119 DNA µg (0.5 OD) (1) 3,162 bp phy3060 PLK DNA (E. coli µg (0.2 OD) >70% RF I (1) 4,870 bp µg/ml Bacillus shuttle vector) Thermus thermophilus HB µg Prepared by phenol/ Contains >20kb Genomic DNA Solution -chloroform; fragments A 260/ MOLECULAR BIOLOGY 2 Nucleic Acids For complete product descriptions and listings of Takara s DNA vectors, ladders, primers, probes, cassettes, linkers and adaptors, see our web site. (1) = purity contains over 70% ds covalently closed circulation I (RFI) (2) =multi-cloning sizes of phsg396 are partially converted sites of phsg

73 DNA Molecular Weight Standard Markers 2MOLECULAR BIOLOGY Nucleic Acids All ladders and digests supplied with 6X loading buffer. Product Product No. Quantity 20 bp DNA Ladder 3409A/B 50 µg 13 fragments (100 lanes) 20 to approx. 500 bp 100, 200 and 500 bp reference bands 100 bp DNA Ladder 3407A 50 µg 11 fragments (100 lanes) 100 to approx. 1,500 bp 500 bp reference band 3407B 100 µg (200 lanes, A 2) 200 bp DNA Ladder 3410A/B 250 µg 13 fragments (100 lanes) 200 to approx. 4,000 bp 1,000 and 2,000 bp reference bands 500 bp DNA Ladder 3411A/B 250 µg 10 fragments (100 lanes) 500 to approx. 5,000 bp 2,000 bp reference band 1 kb DNA Ladder 3412A/B 50 µg 10 fragments (100 lanes) 1 to approx. 10 kb 5,000 bp reference band 2.5 kb DNA Ladder 3413A/B 50 µg 14 fragments (100 lanes) 2.5 to approx 35 kb 10 kb reference band Wide-Range DNA Ladder 3415A 1 ml 16 quantitated fragments (100 lanes) 50 bp to 10 kb 1,400/1,550 bp reference bands фx174 Hae III Digest 3405A 20 µg 11 fragments (100 lanes) 72 to 1,353 bp Use to size fragments <1 kb 3405B 100 µg (500 lanes, A 5) фx174 Hinc II Digest 3406A 20 µg 13 fragments (100 lanes) 79 to 1,057 bp Use to size fragments <1 kb 3406B 100µg (500 lanes, A 5) phy Marker 3404A 20 µg 9 fragments (100 lanes) 80 to 4,870 bp 3404B 100 µg (500 lanes, A 5) λ-ecot14 I Digest µg 11 fragments (100 lanes) 74 to 19,329 bp Use to size fragments from 1 8,000 bp λ-bst P I Digest µg 14 fragments (100 lanes) 117 to 8,453 bp Use to size fragments from 1 5,000 bp λ-hind III Digest µg 8 fragments (100 lanes) 125 to 23,130 bp Use to size fragments from 2 10 kb λ-ecot14 I/Bgl II Digest µg 18 fragments (100 lanes) 60 to 22,010 bp Use to size fragments from 1 20 kb 20 bp DNA Ladder (Dye Plus) 3420A 100 reactions (500 µl) 3420B 200 reactions (500 µl 2) 50 bp DNA Ladder (Dye Plus) 3421A 100 reactions (500 µl) 3421B 200 reactions (500 µl 2) 100 bp DNA Ladder (Dye Plus) 3422A 100 reactions (500 µl) 3422B 200 reactions (500 µl 2) 200 bp DNA Ladder (Dye Plus) 3423A 100 reactions (500 µl) 3423B 200 reactions (500 µl 2) 250 bp DNA Ladder (Dye Plus) 3424A 100 reactions 3424B 200 reactions 500 bp DNA Ladder (Dye Plus) 3425A 100 reactions (500 µl) 3425B 200 reactions (500 µl 2) 1 kb DNA Ladder (Dye Plus) 3426A 100 reactions (500 µl) 3426B 200 reactions (500 µl 2) Wide-Range DNA Ladder 3427A 100 reactions 500 µl 100-2,000 bp 3427B 200 reactions (500 µl 2) 100-2,000 bp Wide-Range DNA Ladder 3428A 100 reactions (500 µl) 100-5,000 bp 3428B 200 reactions (500 µl 2) 100-5,000 bp Note: reference band means a band that is brighter then others 72

74 DNA Molecular Weight Standard Markers Determination of the Molecular Weight of Nucleic Acids Takara offers a variety of DNA ladders suitable for different applications. Several standard l and фx174 digests are offered, as well as 20 bp 2.5 kb DNA ladders. A Wide Range Ladder, which consists of 16 quantitated fragments from 50 10,000 bp, allows both sizing and quantitation over a broad size range. This ladder is premixed with loading dyes and glycerol for direct gel electrophoresis. Supplied 6X Loading Buffer 30% glycerol, 30 mm EDTA, 0.03% Bromophenol Blue, 0.03% Xylene Cylanol Form Cat.# A 10 mm Tris-HCl, ph 8.0, 1 mm EDTA Cat.# 3407A,B 10 mm Tris-HCl, ph 7.4, 1 mm EDTA Cat.# mm Tris-HCl, ph 8.0, 1 mm EDTA, 6% Glycerol, 0.02% Bromophenol Blue, 0.02% Xylene Cyanol, 0.02% NaN 3 Note: Since the terminal fragments of the λ DNA digests contain COS ends, heat treatment is required before use. Denaturation of the fragments may occur unless dilution and heating is performed in TE or TEN buffer. Do not use these markers on polyacrylamide gels. MOLECULAR BIOLOGY Banding Patterns of Takara DNA Ladders 20 bp DNA Ladder (3409A) bp bp DNA Ladder (3407A) bp bp DNA Ladder (3410A) bp bp DNA Ladder (3411A) bp 5,000 4,000 3,000 1,500 1, kb DNA Ladder (3412A) bp 15,000 10,000 5,000 4,000 3,000 2,000 1,000 2 Nucleic Acids kb DNA Ladder (3413A) kb Wide Range DNA Ladder (3415A) bp fx174-hae III Digest (3405A) bp 1,353 1, / fx174-hinc II Digest (3406A) bp 1, / phy Marker (3404A) bp 4,870 2,016 1,360 1,

75 Nucleic 2MOLECULAR Acids BIOLOGY Banding Patterns of Takara DNA Ladders, cont. l-ecot14 I Digest (3401) bp l-bstp I Digest (3402) bp l-hind III Digest (3403) Guideline for Selecting Molecular Weight Markers and by Recommended Gel Concentration bp l-ecot14 I/Bgl II Digest (3408) bp Nucleic Acids Analyzed DNA Size < 500 bp 500 1,000 bp 1,000 5,000 bp >5,000 bp Gel Concentration > 3% 2 3% 0.7 2% < 0.7% l-ecot14 I/Bgl II digest l-ecot14 I/Bgl II digest l-ecot14 I digest l-ecot14 I digest (3408A/B) (3408) (3401) (3401) fx174-hae III digest fx174-hae III digest l-bst P I digest l-bst P I digest (3405A/B) (3405A/B) (3402) (3402) fx174-hinc II digest fx174-hinc II digest l-hin d III digest l-hin d III digest (3406A/B) (3406A/B) (3403) (3403) Appropriate phy Marker phy Marker l-ecot14 I/Bgl II digest l-ecot14i/bgl II digest MW Marker (3404A/B) (3404A/B) (3408) (3408) 20 bp DNA Ladder 20 bp DNA Ladder phy Marker 1 kb DNA Ladder (3409A) (3409A) (3404A/B) (3412A) 100 bp DNA Ladder 100 bp DNA Ladder 200 bp DNA Ladder 2.5 kb DNA Ladder (3407A/B) (3407A/B) (3410A) (3413A) Wide-Range DNA Ladder Wide-Range DNA Ladder 500 bp DNA Ladder Wide-Range DNA Ladder (3415A) (3415A) (3411A) (3415A) Wide-Range DNA Ladder (3415A) 74

76 14-30 ssrna Ladder Marker ssrna Ladder Marker Cat.# µl (25 lanes) An ssrna Molecular Size Marker for Fragments Shorter than 30 Bases in Gel Electrophoresis This marker consists of 5 ssrna fragments between 14 and 30 bases. (Volume: 2.5 µl per well) Fragment size (bases): 30, 26, 22, 18, 14 Form RNase free water 80 C MOLECULAR BIOLOGY Protein Molecular Weight Marker Protein Molecular Weight Marker (Low) Cat.# lanes Protein Molecular Weight Marker (High) Cat.# lanes Protein Molecular Weight Marker (Broad) Cat.# lanes Determination of Protein Molecular Weights Takara s Protein Molecular Weight Markers are designed for use in SDS- Polyacrylamide gel electrophoresis. These markers come in three ranges: Low (Molecular weight range: kda), High (Molecular weight range: kda) and Broad (Molecular weight range: kda). Each protein in these markers is quantitated to yield uniform band intensities when stained with Coomassie Brilliant Blue R-250 after running on an SDS polyacrylamide gel. Five µl of 20-fold diluted marker is recommended per lane of SDS-PAGE minigel for standard use. Component Protein Low (3450) Protein Source M.W. (Da) Phosphorylase B Rabbit muscle 97,200 Serum Albumin Bovine 66,409 Ovalbumin Hen egg white 44,287 Carbonic anhydrase Bovine 29,000 Trypsin Inhibitor Soy bean 20,100 Lysozyme Hen egg white 14,300 Broad (3452) Protein Source M.W. (Da) Myosin Pig 200,000 b-galactosidase E. coli 116,000 Phosphorylase B Rabbit muscle 97,200 Serum Albumin Bovine 66,409 Ovalbumin Hen egg white 44,287 Carbonic anhydrase Bovine 29,000 Trypsin Inhibitor Soy bean 20,100 Lysozyme Hen egg white 14,300 Aprotinin Bovine pancreas 6,500 Concentration 12 µg/µl (Cat.# 3450) 10 µg/µl (Cat.# 3451) 18 µg/µl (Cat.# 3452) Protein Molecular Weight Marker and 1 m DTT: 20 C. 5X Loading Buffer: room temperature after opening. 2 Nucleic Acids High (3451) Protein Source M.W. (Da) Myosin Pig 200,000 b-galactosidase E. coli 116,000 Phosphorylase B Rabbit muscle 97,200 Serum Albumin Bovine 66,409 Ovalbumin Hen egg white 44,

77 Ribonucleoside 5'-Triphosphates 2MOLECULAR BIOLOGY ATP Cat.# µmol GTP Cat.# µmol CTP Cat.# µmol UTP Cat.# µmol Concentration 100 mm Volume 250 µl Purity 95% Deoxyribonucleoside 5'-Triphosphates Form Aqueous solution (lithium salts), ph 7 20 C Note: Dilute with an appropriate buffer, before use. datp Cat.# µmol dgtp Cat.# µmol dctp Cat.# µmol dttp Cat.# µmol Concentration 100 mm Form Aqueous solution (sodium salts), ph 7 to 9 Nucleic Acids Purity 98% 20 C Note: Dilute with an appropriate buffer, before use. dntp Mixture dntp Mixture Cat.# 4030 ea. 3.2 µmol/1.28 ml Ready to use in PCR Reaction Without Dilution General Applied Volume per PCR reaction: g 4 µl for 50 µl PCR with TaKaRa Taq, TaKaRa Ex Taq (sufficient reagent supplied for 320 reactions) g 8 µl for 50 µl PCR with TaKaRa LA Taq (sufficient reagent supplied for 160 reactions) Suitable as Substrate for Other DNA Polymerase Reactions This product is a mixture of datp, dctp, dgtp, dttp (each 2.5 mm). Concentration ea. 2.5 mm Purity ea. 98% PCR Test Validated performance of DNA amplification by Polymerase Chain Reaction (PCR) was confirmed. No contamination with RNase was confirmed. Form Aqueous solution (sodium salts), ph 7 to 9 20 C Volume 1.28 ml 76

78 M13 Primers M13 Primer M1 d(agtcacgacgttgta) Cat.# pmol M13 Primer M2 d(cccagtcacgacgtt) Cat.# pmol M13 Primer RV d(caggaaacagctatgac) Cat.# 3830A 150 pmol M13 Primer RV d(caggaaacagctatgac) Cat.# 3830B 3,000 pmol M13 Primer M3 d(gtaaaacgacggccagt) Cat.# pmol M13 Primer M4 d(gttttcccagtcacgac) Cat.# 3832A 150 pmol M13 Primer M4 d(gttttcccagtcacgac) Cat.# 3832B 3,000 pmol Purity Confirmed by HPLC Primer activity is confirmed by Sanger dideoxy method Intended Usage Available as primer for DNA sequencing of inserts cloned in an M13mp type vector or puc type vector. Form Lyophilized Shipping Room temperature 20 C Related Product M13 Primer RV-N (Cat.# 3833) MOLECULAR BIOLOGY 2 M13 Primer RV-N M13 Primer RV-N d(tgtggaattgtgagcgg) Cat.# pmol Purity Confirmed by HPLC Primer activity is confirmed by Sanger dideoxy method Intended Usage Available as primer for DNA sequencing of inserts cloned into a ptv118n/119n vector. Form Lyophilized Shipping Room temperature 20 C Related Products M13 Primer M1 Cat.# 3810 M13 Primer M2 Cat.# 3820 M13 Primer M3 Cat.# 3831 M13 Primer M4 Cat.# 3832A M13 Primer M4 Cat.# 3832B M13 Primer RV Cat.# 3830A M13 Primer RV Cat.# 3830B Nucleic Acids Ladderman (BcaBEST ) Sequencing Primers Ladderman Sequencing Primer RV-M d(gagcggataacaatttcacacagg) Cat.# pmol Ladderman Sequencing Primer M13-20 d(cgacgttgtaaaacgacggccagt) Cat.# pmol Ladderman Sequencing Primer RV-P d(ggaaacagctatgaccatgattac) Cat.# pmol Ladderman Sequencing Primer T3 d(attaaccctcactaaagggaa) Cat.# pmol Ladderman Sequencing Primer T7 d(taatacgactcactataggg) Cat.# pmol Purity Confirmed by HPLC Primer activity is confirmed by Sanger dideoxy method Intended Usage Primers for DNA sequencing of inserts cloned in an M13mp type vector, puc or pbluescript II type vector. Note: pbluescript is a registered trademark of Stratagene. Form Lyophilized Shipping Room temperature 20 C 77

79 2MOLECULAR BIOLOGY Random Primers Random Primer (hexadeoxyribonucleotide mixture; pd(n)6) Cat.# nmol Random Primer (nonadeoxyribonucleotide mixture; pd(n)9) Cat.# nmol cdna Synthesis Generation of Labeled Probes for Hybridization As a Primer during Synthesis of 1st Strand in RT-PCR These primers are 6 mer, or 9 mer's, of random sequence deoxyribonucleotide mixture (theoretically 4 6 or 4 9 sequences), a phosphorylated 5 -end. The 6 mer pd(n)6 (80 nmol) corresponds to approx.150 µg, 5 OD. Primers that have the 9 mer pd(n)9 (50 nmol) corresponds to approx.150 µg, 5 OD. Form Lyophilized Note: By dissolving pd(n)9 in 1 ml of RNase Free dh 2 O (Final conc. 50 µm), it can be used in Takara's RT-PCR Kits. By dissolving pd(n)6 in 4 ml of RNase Free dh 2 O (Final conc. 20 µm), it can be used in Takara's RT-PCR Kits. Shipping Room temperature 20 C Related Products Ladderman Labeling Kit Cat.# reactions Random Primer DNA Labeling Kit Ver.2 Cat.# reactions BcaBEST RNA PCR Kit Ver. 1.1 Cat.# RR023A 100 reactions BcaBEST RNA PCR Kit Ver. 1.1 Cat.# RR023B 200 reactions RNA PCR Kit Ver.3.0 Cat.# RR019A 100 reactions RNA PCR Kit Ver.3.0 Cat.# RR019B 200 reactions RNA LA PCR Kit (AMV) Ver1.1 Cat.# RR012A 50 reactions mrna Selective PCR Kit Ver.1.1 Cat.# RR025A 50 reactions mrna Selective PCR Kit Ver.1.1 Cat.# RR025B 100 reactions Nucleic Acids Adaptors Adaptor, BamH I (Bgl II) Sma I d(gatccccggg) Cat.# ,000 pmol Adaptor, EcoR I Sma I d(aattcccggg) Cat.# ,000 pmol Adaptor, Hind III Sma I d(agctcccggg) Cat.# ,000 pmol Adaptor, Sal I Sma I d(tcgacccggg) Cat.# ,000 pmol Adaptor, EcoR I Not I BamH I 5 HO AATTCGGCGGCCGCGGATCC GCCGCCGGCGCCTAGG p5 Cat.# ,000 pmol Intended use For introducing cohesive restriction sites into blunt-ended DNA. Sequence BamH I (Bgl II) - Sma I d(gatccccggg) EcoR I - Sma I d(aattcccggg) Hind III - Sma I d(agctcccggg) Sal I - Sma I d(tcgacccggg) Purity Confirmed by HPLC Biological activity is confirmed by self ligation-restriction enzyme cleavage. Note Restriction sites will be lost when Cat.# 4501 and Cat.# 4503 are used for Bgl II and Hind III sites, respectively. Form Lyophilized Shipping Room temperature 20 C 78

80 Phosphorylated Linkers Linker, papa I, phosphorylated pd(ggggcccc) Cat.# 4605P 5,000 pmol Linker, pbam H I, phosphorylated (8mer) pd(cggatccg) Cat.# 4610P 5,000 pmol Linker, pbamh I, phosphorylated (12 mer) pd(cccggatccggg) Cat.# 4810P 5,000 pmol Linker, pbgl II, phosphorylated pd(cagatctg) Cat.# 4621P 5,000 pmol Linker, pcla I, phosphorylated pd(catcgatg) Cat.# 4630P 5,000 pmol Linker, pecor I, phosphorylated (8 mer) pd(ggaattcc) Cat.# 4640P 5,000 pmol Linker, pecor I, phosphorylated (12 mer) pd(ccggaattccgg) Cat.# 4840P 5,000 pmol Linker, phind III, phosphorylated (8 mer) pd(caagcttg) Cat.# 4660P 5,000 pmol Linker, pnco I, phosphorylated (8 mer) pd(gccatggc) Cat.# 4665P 5,000 pmol Linker, pnot I, phosphorylated (8 mer) pd(gcggccgc) Cat.# 4966P 5,000 pmol Linker, pnot I, phosphorylated (10 mer) pd(agcggccgct) Cat.# 4967P 5,000 pmol Linker, psal I, phosphorylated pd(ggtcgacc) Cat.# 4680P 5,000 pmo Linker, psma I, phosphorylated pd(cccggg) Cat.# 4585P 5,000 pmol Linker, pspe I, phosphorylated pd(gactagtc) Cat.# 4686P 5,000 pmol Linker, psse 8387 I (ppst I), phosphorylated pd(cctgcagg) Cat.# 4983P 5,000 pmol Linker, pxba I, phosphorylated pd(ctctagag) Cat.# 4693P 5,000 pmol Linker, pxho I, phosphorylated pd(cctcgagg) Cat.# 4694P 5,000 pmol Intended Use Because 5 -end of these linkers is phosphorylated, they can be directly used in ligations. The cloning of a target gene into a vector can be easily performed matching the structural gene frame by selective use of linkers (8 mer, 10 mer, 12 mer). Purity Confirmed by HPLC Biological activity is confirmed by self ligation and restriction enzyme cleavage Nonphosphorylated Linkers Form Lyophilized Purity Confirmed by HPLC Biological activity is confirmed by self ligation and restriction enzyme cleavage Form Lyophilized Shipping Room temperature Note: These products are single-stranded oligonucleotides. For use in subsequent reactions, dissolve in an appropriate buffer such as TE, incubate at 70 C for 10 min, and then anneal by cooling slowly. 20 C Linker, BamH I (10 mer) d(ccggatccgg) Cat.# 4710A 10,000 pmol Linker, Sal I d(ggtcgacc) Cat.# 4680A 10,000 pmol Shipping Room temperature Note: These products are single-stranded oligonucleotides. For use in the subsequent reaction, dissolve in an appropriate buffer such as TE, incubate at 70 C for 10 min, and then anneal by cooling slowly. 20 C MOLECULAR BIOLOGY 2 Nucleic Acids 79

81 2MOLECULAR BIOLOGY Nucleic Acids Synthetic sirna Quantitation Core Kit Synthetic sirna Quantitation Core Kit Cat.# reactions Kit Components Detects sirna in Total RNA with SYBR Green I Real Time PCR Excellent for Low Copy Number sirnas Terminal Transferase Buffer 390 µl Terminal Transferase 30 µl datp 150 µl Synthetic sirna Quantitation Core Kit is designed to quantify synthetic sirna when Recombinant RNase Inhibitor 30 µl used in combination with real time PCR analysis, and is used to assay the residual Oligo dt Primer µl amount of the administered sirna in total RNA samples extracted from cells, RT Buffer 270 µl tissues, blood, and other specimens. RT Enzyme Mix 30 µl Small interfering RNA (sirna), sometimes also referred to as silencing RNA or short Universal Primer (10 µm) 120 µl EASY Dilution (for Real Time PCR) 1 ml interfering RNA, is double stranded RNA, that is typically nucleotides in Control sirna (20 nm) 10 µl length. sirna can interfere with specific gene expression in cells by hybridizing to Control Primer (10 µm) 20 µl the corresponding mrna, resulting in mrna degradation. 1 Contains enough reagents for 30 reactions of poly da tailing and reverse transcription Synthetic sirna is used as a tool to understand protein function. An sirna is and enough Universal Primer for 120 real time PCRs designed that complements a target gene s mrna sequence. It is introduced into a 2 Specially designed Oligo dt Primer for this product cell, where it will hybridize to the target mrna, resulting in specific mrna degradation. Expression of the corresponding protein is knocked out and its function can be assessed by studying the resulting effect. Synthetic sirna has been tested successfully in several disease models. Synthetic sirna quantitation in cells, tissues and 20 blood plays an important role in sirna drug development. Synthetic sirna generally has two deoxythymidine nucleotides (dtt) as an overhang at the 3 ends. The Synthetic sirna Quantitation Core Kit adds polydeoxyadenine (poly da) to the 3 overhangs and then carries out a reverse transcription reaction with a specific oligo dt primer, allowing preparation of complementary DNA from synthetic sirna molecules. Highly sensitive quantitation of the synthetic sirna can be achieved by using the prepared cdna as template in a SYBR Premix Ex Taq TM Series. Note: This kit is designed to quantify synthetic sirna with deoxythymidines d(tt) as a 3 overhang. For synthetic sirna with 3 overhangs other than d(tt), prepare a specific oligo dt primer with a modified sequence. This product does not work with synthetic sirnas that lack 3 overhangs. 80

82 Random Primer DNA Labeling Kit, Ver. 2 Random Primer DNA Labeling Kit, Ver. 2 Cat.# reactions Application Labeling DNA for Use in Hybridization Probes The Random Primer DNA Labeling Kit, Version 2 is designed to yield DNA probes with high activity for hybridization and can be used to label DNA with [ 32 P]-a-, [ 35 S]-a- or [ 3 H]-a-dCTP. This kit is based on a modified method of Feinberg and Vogelstein that utilizes random oligonucleotide primers and the cloned exonucleasefree Klenow fragment of E. coli DNA polymerase I. The use of longer 9-mer primers and exonuclease-free enzyme results in higher labeling efficiency and longer probes. This method overcomes many of the disadvantages of the conventional nick translation procedure while producing probes from small amounts of DNA (10 to 20 ng). It can also be used to label DNA fragments that are embedded in low-melting temperature agarose gel slices. Ladderman DNA Labeling Kit The Ladderman Labeling Kit is a novel ran dom primer la bel ing system that takes advantage of longer prim ers and a thermostable DNA polymerase iso lat ed from Bacillus caldotenax YT-G (Bca DNA polymerase). Unlike the E. coli Klenow fragment or mod i fied T7 DNA poly merase, thermostable Bca DNA poly merase allows sta ble po ly mer iza tion through templates with high GC con tent (highly structured regions). The longer 9-mer prim ers in crease the efficiency of incorporation of labeled nu cle otides up to 80%. With this kit, random priming is ac com plished within 10 minutes to create efficiently-labeled DNA probes. The kit is designed for use with [ 32 P]-a-, [ 35 S]-a-, or [ 3 H]-a-dCTP labels, but it can also be used with biotin, digoxigenin or DNP-labeled dutps by re place ment of the dntp mixture. Generally, a probe with spe cif ic activity of >10 9 dpm/mg DNA will be obtained with [ 32 P]-a-dCTP (~3,000 Ci/mmol, 111 TBq/mmol). Kit Components 6045 Random Primer (9-mer) 60 µl 10X Buffer 75 µl dntp Mixture 75 µl (0.2 mm each of datp, dgtp and dttp) Exo-free Klenow Fragment (2 U/µL) 30 µl Control DNA (25 ng/ml) 10 µl (λ DNA digested with Hind III, 25ng/µL) Ladderman DNA Labeling Kit Cat.# reactions Application Random Primer Labeling of DNA Using Thermostable Bca DNA Polymerase Kit Components 6046 Random Primer (9-mer) 80 µl 10X Buffer 100 µl dntp Mixture 100 µl (0.2 mm each of datp, dgtp, and dttp) Bca DNA Polymerase (2 U/µL) 40 µl (cloned and modified B. caldotenax DNA polymerase) Control DNA (25 ng/µl) 10 µl (l DNA digested with Hind III, 25ng/µL) MOLECULAR BIOLOGY 2 Nucleic Acids 81

83 PrimeScript Reverse Transcriptase 2MOLECULAR BIOLOGY DNA Labeling and Detection Kits PrimeScript Reverse Transcriptase Cat.# 2680A 10,000 U PrimeScript Reverse Transcriptase Cat.# 2680B 40,000 U (A 4) PrimeScript Reverse Transcriptase Cat.# 2680C 100,000 U (A 10) RT-PCR First Long cdnas and Second-Strand Synthesis Primer Extension and Random Priming for Preparation of DNA Probes PrimeScript Reverse Transcriptase is a novel MMLV-RT-based RTase reverse transcriptase, developed by TaKaRa Bio Inc., that offers robust reverse transcription on any RNA template. PrimeScript RTase is an exceptionally robust RTase which provides complete elongation through RNA templates containing higher-order (i.e., high GC) structures. This enzyme works well on challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. It produces full-length cdnas of up to 12 kb in length with good yield. Concentration 200 units/μl Form 20 mm Tris-HCI (ph7.8),100 mm NaCl, 1 mm EDTA,1 mm DTT, 50% Glycerol(v/v) Source Purified from an E. coli strain expressing a recombinant enzyme. Unit Definition One unit is the amount of the enzyme that incorporates 1 nmol of [ 3 H]dTTP in 10 minutes at 37 C, with poly(ra), oligo(dt) as the primer-template. 1st-Strand cdna Extension Test Using the standard protocol, 1st strand cdna is synthesized by incubating 500 ng of total RNA prepared from mouse heart and 50 pmol of Oligo dt primer with 100 units of PrimeScript Reverse Transcriptase at 42 C for 30 minutes. PCR is then performed with this RT product as a template and yield confirmed using agarose gel electrophoresis. Purity Nuclease activity is not detected under any of the following conditions, demonstrated by the agarose gel electrophoresis pattern: 1. After incubation of 1 μg of λdna-hind III fragments with 200 units of this enzyme for 1 hour at 37 C. 2. After incubation of 1 μg of supercoiled pbr322 DNA with 200 units of this enzyme for 1 hour at 37 C. 3. After incubation of 1 μg of 16S and 23S rrna with 200 units of this enzyme for 1 hour at 37 C. Composition of Supplied Reagent X PrimeScript Buffer (for cdna synthesis) 250 mm Tris-HCl, ph mm KCl 15 mm MgCl 2-20 Related Product PrimeScript 1st Stand cdna Synthesis Kit (Cat.# 6110A/B) Application Example A comparison of PrimeScript RTase and six competitor RTases in firststrand cdna synthesis : PrimeScript 200 U 2: Company A 3: Company B 4: Company C 5: Company D 6: Company E 7: Company F Lane 1 contains the PrimeScript reaction, Lanes 2-6 were tested using the manufactures recommended protocols. PrimeScript shows excellent cdna yield., high sensitivity and low background compared to other enzymes. 82

84 PrimeScript 1st Strand cdna Synthesis Kit Features Excellent Elongation: able to synthesize long cdnas (up to 12 kb) with good yield. Robust and Efficient: carries out reverse transcription at standard RT temperature (42 C), even when RNA template contains high-order structures. The PrimeScript 1st Strand cdna Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cdna from total or poly(a)+ RNA using PrimeScript RTase. PrimeScript RTase is a MMLV-based RTase which possesses excellent elongation ability and is capable of synthesis of cdna of up to 12 kb in length with good yield. In addition, this enzyme works well on even challenging templates at 42 C, making high-temperature transcription, which increases the risk of RNA degradation, unnecessary. First strand cdnas synthesized with this kit can be used for a variety of applications including second strand synthesis, hybridization, PCR amplification, and real-time PCR. Kit Components (50 reactions): #6110A PrimeScript RTase 200 U/μL 5X PrimeScript Buffer RNase Inhibitor 40 U/μL dntp Mixture 10 mm each Oligo dt Primer 50 μm Random 6 mers 50 μm RNase free dh 2 O Not available in the United States PrimeScript 1st Strand cdna Synthesis Kit Cat.# 6110A 100 reactions PrimeScript 1st Strand cdna Synthesis Kit Cat.# 6110B 200 reactions (A 2) Synthesis of Full Length 1st Strand cdna From Total or poly(a)+ RNA 50 μl 200 μl 25 μl 50 μl 50 μl 100 μl 1 ml MOLECULAR BIOLOGY 2 Comparison of Takara s Site-Directed Mutagenesis Systems* Product Mutan -Super Express Km LA PCR in vitro Mutagenesis Kit Mutan -Express Km Cat. # RR022 RR Principle ODA-LA PCR Method** LA PCR Restriction Enzyme Method ODA Method Takara s Mutan -Super Express Km is designed to achieve site-directed mutagenesis in just one day. It is based on Oligonucleotide-directed Dual Amber (ODA) method, and uses LA (Long and Accurate) PCR Amplification. The LA PCR in vitro Mutagenesis Kit is an improved system to introduce a series of site-directed mutations into long DNA fragments cloned in puc or puc-derived vectors containing the puc multiple cloning site. This system takes advantage of LA (Long and Accurate) PCR technology by including the Takara LA Taq enzyme and LA Buffer II. Takara s Mutan -Super Express Km is designed to efficiently introduce site-directed mutations. Using the simplified procedure, a desired mutation can be introduced in 3 days without ssdna isolation. Efficiency >80% >60% 70-95% Vectors pkf18k-2/19k-2 puc18/19 (118/119) pkf18k-2/19k-2 Host Strains sup any strains sup, muts Preparation of No Preparation No Preparation No preparation ssdna Time Required 1 day 2-3 days 3 days Notes Transformation using only PCR products No need for specific host strains No need to prepare ssdna (<2 kb DNA fragments) Quantity 20 reactions 10 reactions 20 reactions * For more information on these products, please visit our website. Cat.# RR022: 80 C cells; 20 C for components Cat.# RR016: 20 C Cat.# 6090: (Enzyme/Oligo Set: 20 C) Sequencing, Mutagenesis and Mutation Analysis 83

85 Deletion Kit for Kilo-Sequencing 2MOLECULAR BIOLOGY Sequencing, Mutagenesis and Mutation Analysis/Molecular Biology Reagents Deletion Kit for Kilo-Sequencing Cat.# reactions Application Sequence Analysis of Long DNA fragments The Deletion Kit for Kilo-Sequencing is designed to aid se quence analysis of long DNA fragments cloned into the mul ti ple cloning sites of M13 phage vectors (mp18/19) or puc-related plasmid or phagemid vectors (puc18/19). The kit is used to create nested unidirectional deletions in the tar get DNA, thereby progressively moving the primer binding site on the vector closer to the sequence of interest. Op ti mized reagent cocktails are included to ensure efficient self-circularization of the deletion subclones. *Ligation solution A and B are provided in DNA Ligation Kit Ver 1 (Cat. # 6021) RNase-Free Water This RNase-Free Water is suitable for use with RNA samples. It is not prepared using hazardous DEPC (diethylpyrocarbonate), and therefore avoids risk of inhibiting RT-PCR which occur with inhibiting residual DEPC. Kit Components 6030 Exo III Buffer 500 µl Exonuclease III (180 U/µL) 10 µl Mung Bean Nuclease Buffer 500 µl Mung Bean Nuclease (25 U/µL) 20 µl Klenow Buffer 250 µl Klenow Fragment (2 U/µL)(large fragment at E.coli DNA polymerase1) 10 µl Ligation Solution A 500 µl Ligation Solution B 60 µl 20 C RNase-Free Water Cat.# tubes 1 ml DNA-OFF Application Elimination of DNA at PCR Workstations DNA-OFF is a non-alkaline, non-corrosive and non-carcinogenic cleansing solution to eliminate DNA contamination and is intended for use at PCR workstations. Contamination of DNA at a PCR work area can result in amplification artifacts. The solution contains a surfactant plus an agent that destroys DNA. DNA-OFF is stable, heat resistant, and ready-to-use. RNase-OFF Elimination of RNase RNase-OFF is a non-alkaline, non-corrosive and non-carcinogenic cleansing solution, which is very active against RNase contamination. It contains a surfactant and an agent inactivating RNase. RNase-OFF is stable, heat resistant and ready-to-use to eliminate RNase from any surface, including the interior of microcentrifuge tubes. Purity Nuclease activity is not detected in any of the following cases, as judged from the agarose gel electrophoresis pattern: RNase activity check: After incubation with 1 μg of total RNA derived from HL60 cells for 16 hours at 37 C DNase activity check: After incubation with 1 μg of λdna for 16 hours at 37 C After incubation with 1 μg of λ-hin d III digest for 16 hours at 37 C After incubation with 1 μg of pbr322 DNA for 16 hours at 37 C 20 C (Shipped at 20 C) DNA-OFF Cat.# ml Kit Components 9036 DNA-OFF Solution 500 ml Room temperature Note: This solution may precipitate when stored at lower temperatures. The precipitate can be brought into solution by incubating at 37 C. RNase-OFF Cat.# ml Kit Components 9037 RNase-OFF Solution 500 ml Room temperature Note: This solution may precipitate when stored at lower temperatures. The precipitate can be brought into solution by incubating at 37 C. 84

86 Summary of TaKaRa Modifying Enzymes Enzyme Quantity Cat.# Page Alkaline Phosphatase, Calf Intestine (CIAP), Cloned 1000 U 2250A U (5 1000) 2250B Alkaline Phosphatase, E. coli (C75) 50 U 2120A U (5 50) 2120B Alkaline Phosphatase, Shrimp 300 U 2660A U 2660B AMV Reverse Transcriptase XL 250 U 2630A 86 BAL 31 Nuclease 50 U 2510A U 2510B Cryonase Cold-Active Nuclease 10,000 U 2670A 93 50,000 U 2670B DNA Ligase (E. coli) 1000 U 2160A U (5 1000) 2160B DNA Polymerase I (E. coli) 500 U 2130A U (5 500) 2130B Recombinant DNase I (RNase-free) 1000 U 2270A U 2270B Exonuclease I 750 U 2650A U 2650B Exonuclease III 5000 U 2170A 92 25,000 U (5 5000) 2170B Klenow Fragment (DNA Polymerase I Large Fragment) 200 U 2140A U* 1000 U 1000 U* 2140AK 2140B 2140BK Mung Bean Nuclease 2000 U 2420A 94 10,000 U (5 2000) 2420B Poly(A) Polymerase 20 U 2180A U 2180B Ribonuclease H (RNase H) 1000 U 2150A U 2150B Ribonuclease Inhibitor 2500 U 2311A/2313A 95 25,000 U Ribonucleoside 5 -Triphosphates ATP 25 µm GTP 25 µm CTP 25 µm UTP 25 µm 2311B/2313B S1 Nuclease 20,000 U 2410A ,000 U (5 20,000) 2410B SP6 RNA Polymerase 3000 U 2520A 90 15,000 U (5 5000) 2520B T4 DNA Ligase 25,000 U 2011A ,000 U 2011B T4 DNA Polymerase 100 U 2040A U 2040B T4 RNA Ligase 1000 U 2050A U 2050B T4 Polynucleotide Kinase 1000 U 2021A U 2021B T7 RNA Polymerase 5000 U 2540A 90 25,000 U 2540B Terminal Deoxynucleotidyl Transferase 300 U 2230A U 2230B Micrococcal Nuclease 15,000 U 2910A 92 * Sequencing grade. For more detailed information, please visit our web site at 76 MOLECULAR BIOLOGY 2 Modifying Enzymes 85

87 AMV Reverse Transcriptase XL 2MOLECULAR BIOLOGY Modifying Enzymes AMV Reverse Transcriptase XL Cat.# 2630A 250 U RT-PCR First- and Second-Strand Synthesis of Long cdnas Primer Extension and Random Priming for Preparation of DNA Probes AMV Reverse Transcriptase (RT) XL synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA-DNA hybrid as a template. The enzyme requires a primer to initiate synthesis and Mg 2+ or Mn 2+ for activity and has 5 g3 ribonuclease H activity. AMV RT XL is highly purified, free of extraneous nucleases and is qualified for use in cdna synthesis using RNA templates up to at least 10 kb in length. It can be used over a temperature range of C, but the maximum yield of full-length cdna product is obtained between C. Tailing Reactions to add Complementary Homopolymer Tails to Vectors and cdna, such as in the Okayama-Berg Method Labeling the 3 -Ends of DNA Fragments using [ 32 P]dNTP or [ 32 P]ddNTP Terminal Deoxynucleotidyl Transferase (TdT) does not require a template for activity, and catalyzes the incorporation of deoxynucleotides into the 3 -OH termini of single- or double-stranded DNA. It requires an oligodeoxynucleotide of at least three bases as a primer. Concentration 7 15 units/µl Form 60 mm potassium phosphate (ph 7.2), 150 mm KCl, 1 mm 2-mercaptoethanol, 50% glycerol Concentration 5 U/µL Terminal Deoxynucleotidyl Transferase Buffer Supplied with 10X RNA PCR Buffer [100 mm Tris-HCl (ph 8.3), 500 mm KCl]. Unit Definition One unit is defined as the amount of AMV RT XL required to catalyze the incorporation of one nmol of [ 3 H] dtmp into acid-insoluble products in 10 minutes at 37 C using poly (ra) oligo(dt) as template-primer. Source Avian Myeloblastosis Virus (Manufactured by Life Science Co.) 80 C. Avoid freeze-thaw cycles. Store at 20 C for up to 6 months. Terminal Deoxynucleotidyl Transferase Cat.# 2230A 300 units Terminal Deoxynucleotidyl Transferase Cat.# 2230B (A 5) 1500 units DNA Topoisomerase I Supplied Buffer 5X TdT Buffer [500 mm HEPES (ph 7.2), 40 mm MgCl 2, 0.5 mm DTT] 0.1% BSA Source E. coli recombinant enzyme Unit Definition One unit is defined as the amount of enzyme that incorporates 1 nmol of [ 3 H]dATP into acid-insoluble products in 1 hour at 37 C and ph 7.2, with calf thymus DNA that is treated with DNase and heat-denatured (activated calf thymus DNA) as the initiator. Purity Endonuclease, Exonuclease and Nickase activity were not detected as judged via gel electrophoresis. 20 C DNA Topoisomerase I Cat.# 2240A 100 units DNA Topoisomerase I Cat.# 2240B (A 5) 500 units Conversion and Analysis of DNA conformation This enzyme has the following four activities: relaxation of supercoiled circular DNA; produces knots in single-stranded, circular DNA and unwinds knots; forms a double-stranded, closed circular DNA from two single-stranded circular DNAs that are complementary to each other; and joins two molecules when a nick exists in one of two double-stranded circular DNAs (catenation), and separates them as well (decatenation). The enzyme is obtained from calf thymus, and is active without Mg 2+, unlike enzymes from procaryotes. Procaryotic DNA Topoisomerase I acts only on negatively supercoiled molecules in reaction 1, but this enzyme can relax both positively and negatively supercoiled molecules. Concentration 5-20 units/μl Form 20 mm potassium phosphate (ph 7.2), 50 mm KCl, 0.05 mm EDTA, 5 mm 2-mercaptoethanol, 0.02% BSA, 50% glycerol Supplied Buffer 10X DNA Topoisomerase I Buffer (350 mm Tris-HCl (ph 8.0), 720 mm KCl, 50 mm MgCl 2, 50 mm DTT, 50 mm spermidine) and 0.1% BSA Unit Definition One unit is the amount of the enzyme that completely relaxes 0.5 μg of supercoiled pbr322 DNA in 50 μl of the reaction mixture in 30 minutes at 37 C. Purity Non-specific Endonuclease activity was not detected as judged from the intact gel electrophoresis pattern 20 C 86

88 Alkaline Phosphatase, Calf Intestinal (CIAP), Cloned Alkaline Phosphatase, Calf Intestinal (CIAP), Cloned Cat.# 2250A 1000 U Alkaline Phosphatase, Calf Intestinal (CIAP), Cloned Cat.# 2250B (A 5) 5000 U Removing Phosphate Groups from 5 -ends of Linear Vectors to Prevent Recircularization during Cloning Dephosphorylation of DNA prior to Kinase Labeling Reactions Calf Intestinal Alkaline Phosphatase (CIAP) catalyzes the hydrolysis of 5 -phosphate groups from DNA and RNA. CIAP is supplied in 10 mm Tris-HCl (ph 8.0), 50 mm KCl, 1 mm MgCl 2, 0.1 mm ZnCl 2 and 50% glycerol. Concentration U/µL Buffer Supplied with 10X Reaction Buffer [500 mm Tris-HCl (ph 9.0), 10 mm MgCl 2 ]. Alkaline Phosphatase, E. coli (C75) Unit Definition One unit is defined as the amount of enzyme that generates 1 µmol/min of p-nitrophenol from p-nitrophenylphosphate at 37 C and ph 9.8. Source Yeast 20 C Reference 1. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, pp 5.72, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MOLECULAR BIOLOGY 2 Alkaline Phosphatase, E. coli (C75) Cat.# 2120A 50 U Alkaline Phosphatase, E. coli (C75) Cat.# 2120B (A 5) 250 U Removing Single Phosphate Groups from 5 -ends of Linear Vectors to Prevent Recircularization during Cloning Dephosphorylation of DNA prior to Kinase Labeling Reactions Bacterial Alkaline Phosphatase nonspecifically catalyzes the removal of most phosphomonoester bonds, but it does not degrade diphosphate or triphosphate linkages. The enzyme is purified from an E. coli strain lacks RNase. Alkaline Phosphatase is supplied in 50 mm Tris-HCl (ph 8.0), 100 mm KCl, 1 mm MgSO 4 and 50% glycerol. Concentration U/µL Buffer Supplied with 10X Reaction Buffer [500 mm Tris-HCl (ph 9.0), 10 mm MgCl 2 ]. Unit Definition One unit is defined as the amount of enzyme that generates 1 µmol/min of p-nitrophenol from p-nitrophenylphosphate at 25 C and ph 8.0. Source E. coli strain C75 20 C References 1. Reid, T.W. and Wilson, I.B. (1971) in The Enzymes 3: Takanami, M. (1967) J. Mol. Biol. 23: Modifying Enzymes Alkaline Phosphatase, Shrimp Alkaline Phosphatase, Shrimp Cat.# 2660A 300 U Alkaline Phosphatase, Shrimp Cat.# 2660B (A 5) 1500 U Application Dephosphorylation of 5 -Terminal Phosphate Groups from DNA Shrimp Alkaline Phosphatase (SAP) nonspecifically catalyzes the dephosphorylation of most phosphate monoesters, but it does not degrade diphosphate or triphosphate linkages. It is irreversibly inactivated by heat denaturation at 65 C for 15 minutes. Concentration 1 U/µL Buffer Supplied with 1mL 10X SAP Buffer [500 mm Tris-HCl (ph 9.0), 50 mm MgCl 2 ]. Unit Definition One unit is the amount of enzyme that produces 1 µmol/min of p-nitrophenol per minute at 37 C and ph 9.6 using p-nitrophenylphosphate as the substrate. Source Shrimp (Pandalus borealis) 20 C Reference 1. Rangnar, L. et al. (1991) Comp. Biochem. Physiol. 99B(4):

89 DNA Ligase (E. coli) 2MOLECULAR BIOLOGY Modifying Enzymes DNA Ligase (E. coli) Cat.# 2160A 1000 U DNA Ligase (E. coli) Cat.# 2160B (A 5) 5000 U Application Joining Two Fragments of DNA with Cohesive Ends E. coli DNA Ligase catalyzes the formation of phosphodiester bonds between doublestranded DNA fragments with 3'-OH and 5'-phosphate ends, in the presence of NAD + as the cofactor. Unlike T4 DNA Ligase, E. coli DNA Ligase can only ligate DNA fragments with cohesive ends under standard reaction conditions. E. coli DNA Ligase is supplied in 10 mm potassium phosphate (ph 7.5), 50 mm KCl, 1 mm DTT, 1 mm EDTA and 50% glycerol. T4 DNA Ligase Joining Two Fragments of DNA (blunt-ended or with compatible overhangs) Adding Linkers to Blunt-Ended DNA T4 DNA Ligase catalyzes the formation of phosphodiester bonds between doublestranded DNA fragments with 3'-OH and 5'-phosphate ends, in the presence of ATP. T4 DNA Ligase is supplied in 10 mm Tris-HCl (ph 7.5), 50 mm KCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. Buffer Supplied with 10X T4 DNA Ligase Buffer [660 mm Tris-HCl (ph 7.6), 66 mm MgCl 2, 100 mm DTT and 1 mm ATP]. Concentration U/µL Unit Definition One unit is defined as the amount of enzyme that ligates >90% of 6 µg of DNA-Hind III fragments in a 20 µl reaction volume in 30 minutes at 16 C. Source Recombinant E. coli 20 C Reference 1. Panasenko, S.M. et al. (1978) J. Biol. Chem. 253: T4 DNA Ligase Cat.# 2011A 25,000 U (200 Weiss units) T4 DNA Ligase Cat.# 2011B (A 25,000 U) 125,000 U (1000 Weiss units) Concentration 350 U/µL (2.8 Weiss units/µl) Unit Definition One ligation unit is defined as the amount of enzyme that ligates >90% of 6 µg of l DNA-Hind III fragments in a 20 µl reaction volume in 30 minutes at 16 C. One unit of the enzyme corresponds to Weiss units by the ATP-PP i exchange reaction in 66 mm Tris-HCl (ph 7.6), 6.6 mm MgCl 2, 10 mm DTT, 66 µm ATP and 3.3 µm [ 32 P] Na 4 P 2 O 7. Source Recombinant E. coli strain 20 C T4 RNA Ligase T4 RNA Ligase Cat.# 2050A 1000 U T4 RNA Ligase Cat.# 2050B (A 5) 5000 U Ligation of Single-Stranded RNA or DNA 3 -end Labeling of Single Stranded RNA or DNA T4 RNA Ligase catalyzes the formation of phosphodiester bonds between singlestranded nucleic acids with 5'-phosphate and 3'-hydroxyl termini. The enzyme requires ATP for activity. The rate of activity is highest for RNA-RNA ligation, lower for RNA-DNA ligation and very low for DNA-DNA ligation. T4 RNA Ligase is supplied in 20 mm Tris-HCl (ph 7.5), 50 mm NaCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. Buffer Supplied with 10X T4 RNA Buffer [500 mm Tris-HCl (ph 7.5), 100 mm MgCl 2, 100 mm DTT and 10 mm ATP]. Concentration U/µL Unit Definition One unit is defined as the amount of enzyme that converts 1 pmol of [5-32 P]pCp into its acid-insoluble form in 10 minutes at 5 C, using oligo(a) as the substrate, labeling of 3 terminus of RNA. Source E. coli B strain infected with T4 am N82 20 C References 1. Romaniuk, P.J. and Uhlenbeck, O.C. (1983) Methods Enzymol. 100: England, T.E. et al. (1980) Methods Enzymol. 65:

90 Poly(A) Polymerase Poly(A) Polymerase Cat.# 2180A 20 U Poly(A) Polymerase Cat.# 2180B (A 5) 100 U Adding poly(a) Tails to RNA Labeling the 3 -End of RNA Poly(A) Polymerase catalyzes the incorporation of adenine residues into the 3'-termini of RNA. The enzyme uses single-stranded RNA as a primer. Poly(A) Polymerase is supplied in 25 mm Tris-HCl (ph 7.9), 500 mm NaCl, 1 mm EDTA, 0.1 mm DTT and 50% glycerol. Concentration U/µL Unit Definition One unit is defined as the amount of enzyme that incorporates 1 nmol of AMP into trna in 10 minutes at 37 C and ph 7.9, using ATP as the substrate. Source E. coli B 20 C References 1. Krug, M.S. and Berger, S.L. (1987) Methods Enzymol. 152: Winter, G. and Brownlee, G.G. (1978) Nucleic Acids Res. 5: Klenow Fragment (DNA Polymerase I Large Fragment) Klenow Fragment (DNA Polymerase I Large Fragment) Cat.# 2140A 200 U Klenow Fragment (DNA Polymerase I Large Fragment) Cat.# 2140B (A 5) 1000 U Klenow Fragment (DNA Polymerase I Large Fragment) Cat.# 2140AK 200 U* Klenow Fragment (DNA Polymerase I Large Fragment) Cat.# 2140BK (AK 5) 1000 U* Random-Primed Labeling Second-Strand cdna Synthesis Dideoxy DNA Sequencing Klenow Fragment (DNA Polymerase I Large Fragment) possesses the 5 g3 polymerase and 3 g5 exonuclease activities of intact DNA Polymerase I, but lacks its 5 g3 exonuclease activity. Klenow Fragment is supplied in 50 mm potassium phosphate (ph 6.5), 1 mm DTT, and 50% glycerol (2140A/B/S) or ethylene glycol (2140AK/BK). Concentration 2-5 U/µL (2140A/B) 2 U/µL (2140AK/BK) *Sequencing grade Buffer Supplied with 10X Reaction Buffer [100 mm Tris-HCl (ph 7.5), 70 mm MgCl 2 and 1 mm DTT]. Unit Definition One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble products in 30 minutes at 37 C and ph 7.4, using poly d(a-t) as the template-primer. Source Recombinant E. coli 20 C MOLECULAR BIOLOGY 2 Modifying Enzymes 89

91 2MOLECULAR BIOLOGY Modifying Enzymes SP6 RNA Polymerase SP6 RNA Polymerase Cat.# 2520A 3000 U SP6 RNA Polymerase Cat.# 2520B (A 5) 15,000 U Application Synthesis of RNA Transcripts SP6 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits a high specificity for bacteriophage SP6 promoter sequences. The enzyme can incorporate labeled or unlabeled nucleotide triphosphates into an RNA transcript. Large quantities of RNA can be synthesized from a DNA sequence cloned downstream from an SP6 promoter. SP6 RNA Polymerase is supplied in 10 mm potassium phosphate (ph 7.9), 150mM NaCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. Concentration U/µL Buffer Supplied with 10X Reaction Buffer [400 mm Tris-HCl (ph 7.5), 60 mm MgCl 2 and 20 mm spermidine], 100 mm DTT and 0.1% BSA. T7 RNA Polymerase T7 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits a high specificity for bacteriophage T7 promoter sequences. The enzyme can incorporate labeled or unlabeled nucleotide triphosphates into an RNA transcript. Large quantities of RNA can be synthesized from a DNA sequence cloned downstream from a T7 promoter. T7 RNA Polymerase is supplied in 20 mm potassium phosphate (ph 7.9), 100 mm NaCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. Concentration U/µL Buffer Supplied with 10X Reaction Buffer [400 mm Tris-HCl (ph 8.0), 80 mm MgCl 2 and 20 mm spermidine] and 50 mm DTT. Unit Definition One unit is defined as the amount of enzyme that incorporates 1 nmol of [ 3 H] GMP using a linear DNA template into acid-insoluble material in 1 hour at 37 C, ph 7.5. Source Recombinant E. coli 20 C Related Products Ribonucleoside 5 -Triphosphates (Cat.# ) T7 RNA Polymerase Cat.# 2540A 5000 U T7 RNA Polymerase Cat.# 2540B (A 5) 25,000 U Application Synthesis of RNA Transcripts Unit Definition One unit is defined as the amount of enzyme that converts 1 nmol of [ 3 H] GMP using a linear DNA template into acid-insoluble material in 1 hour at 37 C and ph 8.0. Source Recombinant E. coli 20 C Related Products Ribonucleoside 5'-Triphosphates (Cat.# ) 90

92 DNA Polymerase I (E. coli) DNA Polymerase I (E. coli) Cat.# 2130A 500 U DNA Polymerase I (E. coli) Cat.# 2130B (A 5) 2500 U DNA Labeling by Nick Translation Producing Blunt Ends from 5 - and 3 -Overhangs Second-Strand Synthesis of cdna DNA Polymerase I catalyzes the incorporation of nucleotides into double-stranded DNA in a 5 g3 direction. It also possesses a 3 g5 exonuclease (proofreading) activity. The 5 g3 exonuclease activity is useful for labeling DNA by nick translation. DNA Polymerase I is supplied in 50 mm potassium phosphate (ph 6.5), 1 mm DTT and 50% glycerol. Concentration 3-6 U/µL Buffer Supplied with 10X Reaction Buffer [500 mm Tris-HCl (ph 7.8), 100 mm MgCl 2, 1 mm DTT and 0.025% (w/w) BSA]. Unit Definition One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble products in 30 minutes at 37 C and ph 7.4, using poly d(a-t) as the template-primer. Source Recombinant E. coli 20 C References 1. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, pp , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2. Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2: Rigby, P.W. et al. (1977) J. Mol. Biol. 113: MOLECULAR BIOLOGY 2 T4 DNA Polymerase T4 DNA Polymerase Cat.# 2040A 100 U T4 DNA Polymerase Cat.# 2040B (A 5) 500 U Creating Blunt Ends from Protruding Ends in Double-Stranded DNA Fragments 3 -End Labeling of Double-Stranded DNA Site-Directed Mutagenesis T4 DNA Polymerase catalyzes the incorporation of nucleotides into double-stranded DNA in a 5'g3' direction. It possesses a strong 3'g5' exonuclease (proofreading) activity, but does not exhibit 5'g3' exonuclease activity. T4 DNA Polymerase is supplied in 200 mm potassium phosphate (ph 6.5), 1 mm DTT and 50% glycerol. Concentration 2-5 U/µL Unit Definition One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble products in 30 minutes at 37 C and ph 8.8, using heat-denatured calf thymus DNA as the template-primer. Source Recombinant E. coli 20 C References 1. Kunkel, T.A. et al. (1987) Methods Enzymol. 154: Deen, K. C. et al. (1983) Anal. Biochem. 135: Wartell, R.M. and Reznikoff, W. S. (1980) Gene 9: Modifying Enzymes Buffer Supplied with 10X Reaction Buffer [330 mm Tris-acetate (ph 7.9), 660 mm potassium acetate, 100 mm magnesium acetate and 5 mm DTT] and 0.1% BSA. 91

93 Micrococcal Nuclease 2MOLECULAR BIOLOGY Micrococcal Nuclease Cat.# 2910A 15,000 U Digestion of Chromatin for Nucleosome Preparation Digestion of Nucleotides included in Crude Cell Extract Micrococcal Nuclease is an endonuclease that preferentially digests single-stranded DNA or RNA, especially at AT- or AU-rich region, but will also digest double-stranded DNA or RNA. This enzyme digests 5 -phosphodiester bonds of DNA and RNA, and yields 3 -phosphate mononucleotides and oligonucleotides. It is commonly used for preparation of nucleosomes during epigenetic analysis studies. This enzyme requires Ca 2+ and is completely inactivated by EDTA or EGTA. Concentration units/µl Form 50 mm Tris-HCl (ph 8.0), 10 mm 2-mercaptoethanol, 50 mm NaCl, 50% glycerol Supplied Buffer 10X Micrococcal Nuclease [200 mm Tris-HCl (ph 8.0), 50 mm NaCl, 25 mm CaCl 2 ] Source Staphylococcus aureus Unit Definition One unit is the amount of enzyme that produce 1 OD 260 of acid-soluble products in 30 minutes at 37 C and ph 8.0, with heat-denatured calf thymus DNA as the substrate. Purity Nuclease activity is not detected after incubation of 1 µg ldna with 50 units of this enzyme for 10 minutes at 37 C in the reaction enzyme buffer (1 H buffer), as judged from the agarose gel electrophoresis pattern. This enzyme is more than 95% homogeneous as judged by SDS-PAGE. 20 C Exonuclease I Exonuclease I Cat.# 2650A 750 U Exonuclease I Cat.# 2650B (A 5) 3750 U Modifying Enzymes Removal of ssdna Fragments in a Reaction Mixture Digestion of Remaining Primers in a PCR Reaction Mixture after Cycling E. coli Exonuclease I is a 3 g5 exonuclease specific for single-stranded DNA, which results in the production of 5 -phosphate mononucleotides. This enzyme has a strict specificity for single-stranded DNA and does not react with double-stranded DNA or RNA. It is inactivated by heat treatment at 80 C for 15 minutes. Buffer Supplied with 1 ml 10X Exonuclease I Buffer [670 mm Glycine-KOH, ph 9.5, 10 mm DTT, 67 mm MgCl 2 ]. Concentration 5 U/µL Unit Definition One unit is the amount of enzyme that produces 10 nmol of acid-soluble products in 30 minutes at 37 C and ph 9.5 using heat-denaturated calf thymus DNA as the substrate. Source Escherichia coli JM1019 carrying plasmids encoding the gene for E. coli Exonuclease I 20 C Exonuclease III Exonuclease III Cat.# 2170A 5000 U Exonuclease III Cat.# 2170B (A 5) 25,000 U Preparation of Partially Single-Stranded DNA for Sequencing Producing Nested Deletions in Double-Stranded DNA Analyzing DNA-Protein Interactions Exonuclease III is a 3 g5 exonuclease that is specific for double-stranded DNA or DNA-RNA hybrids. It will degrade double-stranded DNA with a blunt end, a 5 -overhang, or a nick, but it will not degrade DNA with a 3 -overhang of at least 4 bases. Exonuclease III is supplied in 25 mm Tris-HCl (ph 8.0), 50 mm KCl, 0.5 mm DTT and 50% glycerol. Buffer Supplied with 10X Reaction Buffer [500 mm Tris-HCl (ph 8.0), 50 mm MgCl 2 and 10 mm DTT. Concentration U/µL Unit Definition One unit is defined as the amount of enzyme that produces 1 nmol of acid-soluble products in 30 minutes at 37 C and ph 8.0, with restriction-digested calf thymus DNA as substrate. Source Recombinant E. coli 20 C (when stored for more than 6 months, it should be kept frozen at 80 C) References 1. Wu, C. (1985) Nature 317: Guo, L.H. and Wu, R. (1983) Methods Enzymol. 100:

94 BAL 31 Nuclease BAL 31 Nuclease Cat.# 2510A 50 U BAL 31 Nuclease Cat.# 2510B (A 5) 250 U Application Generating Deletions from Both Ends of DNA Fragments BAL 31 Nuclease has two types of activity. It acts as an exonuclease, degrading double-stranded DNA and RNA from both 5 - and 3 -ends. It also possesses singlestrand endonuclease activity. BAL 31 Nuclease is supplied in 20 mm Tris-HCl (ph 8.0), 100 mm NaCl, 5 mm CaCl 2, 5 mm MgCl 2, 1 mm EDTA and 50% glycerol. Concentration 1-3 U/µL Source Alteromonas espejiana BAL 31 Cryonase Cold-Active Nuclease Buffer Supplied with 2X Reaction Buffer [40 mm Tris-HCl (ph 8.0), 1200 mm NaCl, 24 mm MgCl 2, 24 mm CaCl 2 and 2 mm EDTA]. Unit Definition One unit is defined as the amount of enzyme that produces 1 µg of acid-soluble products from heat-denatured calf thymus DNA in 1 minute at 30 C and ph C Reference 1. Gray, Jr., H.B. et al. (1981) Gene Amplif. Anal. 2: Cryonase Cold-Active Nuclease Cat.# 2670A 10,000 U Cryonase Cold-Active Nuclease Cat.# 2670B (A 5) 50,000 U Application Digestion of all Types of RNA and DNA Cryonase Cold-Active Nuclease is a recombinant endonuclease originating from a psychrophile, Shewanella sp., which is expressed in and purified from E. coli. This enzyme can digest all types of DNA and RNA (single-stranded, double-stranded, linear or circularized), even on ice. Cryonase is useful for digestion of nucleic acids in samples including heat-labile substances such as proteins. Unit Definition One unit is the amount of the enzyme that increases the absorbance at 260 nm by per minute at 37 C and ph 7.5, with salmon sperm DNA as a substrate. Source Purified from E. coli Purity Protease activity is lower than the limit of detection. The contamination of E. coli genome DNA is less than the limit of detection. Form 10 mm Tris-HCl, ph mm NaCl 2 mm MgCl 2 50% glycerol 20 C MOLECULAR BIOLOGY 2 Modifying Enzymes Recombinant DNase I (RNase-free) Recombinant DNase I (RNase-free) Cat.# 2270A 1000 U Recombinant DNase I (RNase-free) Cat.# 2270B (A 5) 5000 U Digestion of Template DNA after in vitro Transcription with T7 or SP6 RNA Polymerase Digestion of Genomic DNA before RT-PCR Nick Translation with DNA Polymerase Construction of a Shotgun DNA Library in the Presence of Mn 2+ DNase Foot Print Analysis of DNA Protein Interaction Recombinant DNase I (RNase-free) is an endonuclease that catalyzes, to the same degree, the random degradation of both single- and double-stranded DNA and produces 5 -P terminal oligonucleotides. This enzyme does not exhibit protease activity. This enzyme is stable around its optimum neutral ph range and is suitable for RNA preparation at neutral ph. Supplied Buffer (10X) 400 mm Tris-HCl, ph mm MgCl 2 50 mm DTT Unit Definition One unit is the amount of enzyme that increases the absorbance at 260 nm by per minute at 25 C, ph 5.0, with calf thymus DNA as the substrate (Kunitz unit). Source Recombinant enzyme. 20 C Concentration 5 U/µL 93

95 Mung Bean Nuclease 2MOLECULAR BIOLOGY Mung Bean Nuclease Cat.# 2420A 2000 U Mung Bean Nuclease Cat.# 2420B (A 5) 10,000 U Creating Blunt Ends from Protruding Ends in Double-Stranded DNA Fragments Digestion of Single-Stranded Regions in Duplex DNA or RNA-DNA Hybrids Mung Bean Nuclease digests single-stranded nucleic acids (both DNA and RNA). If used at very high concentrations, it is capable of degrading double-stranded DNA fragments. Mung Bean Nuclease is supplied in 10 mm Tris-HCl (ph 7.5), 0.1 mm zinc acetate and 50% glycerol. Concentration U/L Buffer Supplied with 10X Reaction Buffer [300 mm sodium acetate (ph 5.0), 1000 mm NaCl, 10 mm zinc acetate and 50% glycerol]. Unit Definition One unit is defined as the amount of enzyme that converts 1 µg of heat-denatured calf thymus DNA into acid-soluble products in 1 minute at 37 C and ph 5.0. Source Mung bean sprouts 20 C References 1. Sheflin, L.G. and Kowalski, D. (1985) Nucleic Acids Res. 13: Kowalski, D. et al. (1976) Biochemistry 15: Kroeker, W.D. et al. (1976) Biochemistry 15: Ribonuclease H (RNase H) Modifying Enzymes Ribonuclease H (RNase H) Cat.# 2150A 1000 U Ribonuclease H (RNase H) Cat.# 2150B (A 5) 5000 U Removal of RNA during Second-Strand cdna Synthesis Removal of Poly(A) Sequences from mrna Ribonuclease H is an endoribonuclease that specifically degrades the RNA strand in an RNA-DNA hybrid. Ribonuclease H is supplied in 25 mm Tris-HCl (ph 7.5), 30 mm NaCl, 0.5 mm EDTA, 1 mm DTT and 50% glycerol. Concentration U/µL Unit Definition One unit is defined as the amount of enzyme that produces 1 nmol of acid-soluble products in 20 minutes at 30 C and ph 7.7, with poly(ra)-poly(dt) as substrates. Source Recombinant E. coli 20 C Reference 1. Vournakis, J.N. et al. (1975) Proc. Natl. Acad. Sci. USA 72: S1 Nuclease S1 Nuclease Cat.# 2410A 20,000 U S1 Nuclease Cat.# 2410B (A 5) 100,000 U Creating Blunt Ends from Protruding Ends in Double-Stranded DNA Fragments Digestion of Single-Stranded Regions in Double-Stranded DNA Mapping RNA Transcripts (Nuclease Protection Assays) S1 Nuclease specifically degrades single-stranded nucleic acids, including singlestranded regions of duplex DNA or RNA. S1 Nuclease is supplied in 10 mm sodium acetate (ph 4.6), 150 mm NaCl, 0.05 mm ZnSO 4 and 50% glycerol. Concentration U/µL Buffer Supplied with 10X Reaction Buffer [300 mm sodium acetate (ph 4.6), 2.8 M NaCl and 10 mm ZnSO 4 ]. Unit Definition One unit is defined as the amount of enzyme that converts 1 µg of heat-denatured calf thymus DNA into acid-soluble products in 1 minute at 37 C and ph 4.6. Source Aspergillus oryzae 20 C References 1. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, pp , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2. Berk, A.J. and Sharp, P.A. (1978) Proc. Natl. Acad. Sci. USA 75:

96 Recombinant Ribonuclease Inhibitor Recombinant Ribonuclease Inhibitor Cat.# 2313A 5000 U Recombinant Ribonuclease Inhibitor Cat.# 2313B 25,000 U cdna Synthesis (Ribonuclease Inhibitor, 0.5 units/µl reaction) In vitro Translation (Ribonuclease Inhibitor, 1 unit/µl reaction) In vitro Transcription with Cell-Free Extract (Ribonuclease Inhibitor, 20 units/µl reaction) In vitro Transcription with SP6 or T7 RNA Polymerase (Ribonuclease Inhibitor, 1 unit/µl reaction) Polysome Isolation (Ribonuclease Inhibitor, 1,000 units/ml reaction) Recombinant RNase Inhibitor forms a 1:1 complex with RNase A and inhibits RNase activity. This reaction is reversible, and ribonuclease activity can be recovered by dissociating the complex with urea or sulfhydryl reagent. This reaction irreversibly inactivates the inhibitor. Recombinant RNase Inhibitor can be added directly to reaction mixtures containing RNA. In addition, because it is a protein, it differs from other competitive inhibitors (nucleotides and inorganic phosphates) by being able to be easily removed from the reaction system using phenol extraction. The inhibitor does not inhibit RNase H activity. The Recombinant RNase Inhibitor is a recombinant protein and is purified from E. coli using affinity chromatography. The activity of this enzyme is similar to RNase inhibitors from porcine liver and human placenta and it can be used in the same applications as those from human placenta and porcine liver RNase inhibitors. Concentration 40 units/µl Unit Definition One unit is the amount of the inhibitor required to inhibit by 50% the activity of 5 ng of RNase A. This inhibitor activity is determined by its ability to inhibit the hydrolysis of cyclic 2, 3 -CMP by RNase A. Source E. coli 20 C MOLECULAR BIOLOGY 2 Ribonuclease Inhibitor (porcine liver) Ribonuclease Inhibitor (porcine liver) Cat.# 2311A 5000 U Ribonuclease Inhibitor (porcine liver) Cat.# 2311B 25,000 U The Porcine Liver RNase Inhibitor is purified from porcine liver by affinity chromatography on an immobilized RNase A column. The character of this enzyme is similar to human placenta, RNase inhibitor. It forms a 1:1 complex with RNase A, and inhibits RNase activity noncompetitively (Ki= M). The inhibitor does not inhibit RNase H activity. The Recombinant RNase Inhibitor is a recombinant protein and is purified from E. coli using affinity chromatography. The activity of this enzyme is similar to RNase inhibitors from porcine liver and human placenta and it can be used in the same applications as those from human placenta and porcine liver RNase inhibitors. Concentration 40 units/µl Unit Definition One unit is the amount of the inhibitor required to inhibit by 50% the activity of 5 ng of RNase A. This inhibitor activity is determined by its ability to inhibit the hydrolysis of cyclic 2, 3 -CMP by RNase A. Source Porcine liver 20 C Modifying Enzymes T4 Polynucleotide Kinase T4 Polynucleotide Kinase Cat.# 2021A 1000 U T4 Polynucleotide Kinase Cat.# 2021B 5,000 U 5'-end-labeling of Double-Stranded DNA 5'-end-labeling of Oligonucleotides or RNA T4 Polynucleotide Kinase catalyzes the transfer of the g-phosphate of ATP to the 5'-hydroxyl-terminus of DNA or RNA, or the 5'-phosphoterminus of DNA or RNA using the exchange reaction. T4 Polynucleotide Kinase is supplied in 50 mm Tris- HCl (ph 7.5), 50 mm KCl, 1 mm DTT, 0.1 μm ATP and 50% glycerol. Concentration 10 units/µl Unit Definition One unit is defined as the amount of enzyme that incorporates 1 nmol of [32P] from [g-32p]atp into acid-insoluble products in 30 minutes at 37 C and ph 7.6, using calf thymus DNA activated by micrococcal nuclease as the substrate. Source Recombinant E. coli 20 C Buffer Supplied with 10X Reaction Buffer [500 mm Tris-HCl (ph 8.0), 100 mm MgCl 2 and 50 mm DTT]. 95

97 2MOLECULAR BIOLOGY Restriction Enzymes takara restriction enzymes Takara Bio Inc. is a world class supplier of life science research products headquartered in Otsu, Shiga, Japan. Takara Bio was the first manufacturer to introduce restriction enzymes to the Japanese market in 1979, and has consistently developed reliable, innovative life science technologies and products. Takara Bio offers a complete line of Restriction and Modifying Enzymes, including our 100 Restriction Enzymes, several of which are unique. Takara s Restriction Enzymes are each supplied with an optimized buffer that provides maximum activity in restriction digests. Quality Control of Restriction Enzymes Takara follows a thorough QC process when manufacturing restriction enzymes. Takara performs several RNase, Protease, and DNase tests, as well as activity and nicking tests. Takara performs four additional quality tests. These tests include the Overdigestion test and the Ligation-Recutting Test. Some restriction enzymes are also analyzed by the pkf3 Cloning Test and the genomic DNA Analysis. Purity Test The Overdigestion Test and the Ligation-Recutting Test are performed on all enzymes while the Genomic DNA Analysis Test and pkf3 Cloning Test are performed on selected enzymes. 1. Overdigestion Test. 1 μg of substrate (usually λ DNA) and excessive amounts of enzyme are incubated for 24 hours. Then, nonspecific DNase is checked by agarose gel electrophoresis. 2. Ligation-Recutting Test. Substrate DNA is digested with a 2 to 50-fold excess of enzyme. Digested DNAs were collected and dissolved in T4 DNA Ligase buffer [66 mm Tris-HCL (ph 7.6), 6.6 mm MgCl 2, 10 mm DTT, 0.4 mm ATP] to obtain a 0.1 to 1.0 um concentration of 5 -terminus. An appropriate amount of T4 DNA Ligase is added and incubated for 1 hour or for hours at 16 C. Collected DNAs are dissolved in the restriction enzyme reaction mixture and recut by the same enzyme. Contamination with ligase inhibitor, phosphatase and exonuclease are determined from the test results. 3. Genomic DNA analysis grade restriction enzyme. When cutting various genomic DNAs with restriction enzymes for genome analysis studies, there is a wide variation of suitable restriction enzymes depending on the species to be analyzed. Takara selects suitable restriction enzymes for various genomic DNA analyses, cuts appropriate bacterial genomic DNA (embedded in agarose; 0.5 μg DNA/50 μl gel) with each lot of enzymes, and performs pulse-field electrophoresis to confirm DNA banding patterns. The amount of enzymes required for complete digestion of bacterial genomic DNA is measured. To determine the minimum amount of enzyme required for complete digestion, 5, 10, 20 and 50 units of each enzyme was added and incubated for 5 and 20 hours. Genomic DNA Analysis Test Tests are performed on selected restriction enzymes (as indicated on product literature). 20 to 150 units of restriction enzyme is added to the appropriate bacterial genomic DNA (embedded in agarose, 0.5 μg DNA/50 μl gel). After 24 hours incubation, pulse-field electrophoresis is performed to confirm DNA banding pattern. 4. pkf3 Cloning Test. Tests are performed on the restriction enzymes that have a cleavage site in the MCS of the Enforcement Cloning System Vector pkf3 DNA. pkf3 DNA is incubated with a 10-fold excess of restriction enzyme. After an inactivation step, digested DNA is treated with a DNA Ligation Kit, Ver. 1 (Cat.# 6021) for 30 minutes at 16 C. TH2 competent cells are transformed using an aliquot of the reaction mixture and cultured on two kinds of plates (LB-Cm-Sm, LB-Cm) for 2 days at 37 C. Trace amounts of exonuclease are assessed by the presence of colonies on the LB-Cm-Sm plates. rpsl + Sm S + Cm R rpsl+ Restriction Enzyme Ligation Transformation Changes into Sm sensitive without exonuclease pkf3 DNA cat-5 (Cm R ) rpsl - rpsl + Sm R + Cm R Host genome E. coli TH2 (Sm resistant) Remains Sm resistant with exonuclease Principle of pkf3 Cloning Test 96

98 Restriction enzymes are currently classified into three types depending on cofactor requirements and cleavage site characteristics. Besides cleaving at their defined restriction sites, Type II restriction enzymes can also exhibit a wide variety of relaxed specificity cleavage or star activity depending on the substrate DNA and reaction conditions. These factors should be considered prior to digestion in order to obtain specific cleavage (see below). Types Cofactors Cleavage Site Enzymes Type I ATP, AdoMet, Mg 2+ Recognition sites and cleavage sites are different and cleavage Eco B, Eco K sites are not fixed. Type II Mg 2+ Cleave DNAs at the recognition sequence or at a defined Eco R I, Bam H I distance from the recognition site. Type III ATP, Mg 2+ Cleave DNAs at fixed sites, though their recognition sites and Eco P I, Hin f III cleavage sites are different. MOLECULAR BIOLOGY Effects of DNA Methylation DNA prepared from bacterial hosts possessing DNA methylase activities will be partially methylated. Such partially methylated DNA may be incompletely digested by methylation-sensitive restriction enzymes. Methylation sites vary depending on substrate DNA and host bacteria. For example, in E. coli, the commonly-used strains C600, HB101 and JM109 contain both Dam and Dcm methylases. In addition, most of the CG sequences in animal-derived DNA are modified to 5m CG, while the CG and CNG sequences in plant DNA derived are generally modified to 5m CG and 5m CNG, respectively. For further information, please refer to the table The effect of methylation on restriction enzyme activity on page 212. GATC CC(A/T)GG Dam methylase Dcm methylase G 6m ATC C 5m C(A/T)GG Star Activity Most restriction enzymes possess star activity a relaxation of their recognition sequence specificity under certain reaction conditions, This activity results in additional DNA cleavage at secondary sites, as well as potentially partial cleavage or DNA nicking activity. Almost all restriction enzymes possess some star activity, though its occurrence is dependent on the enzyme, substrate DNA, and reaction conditions. Star activity can be suppressed by performing reactions at low glycerol and high salt concentrations and neutral ph, even though these conditions may lower general enzyme activity. For further information, please refer to the table The star activities of restriction enzymes on page 209. Partial Cleavage If a substrate DNA is not efficiently cleaved a restriction enzyme with sites in that template, several factors should be considered, including: methylation, star activity, reduced enzyme activity, DNA purity, reaction inhibitors and/or substrate DNA species. In particular, DNAs of differing sizes and/or containing differing number of cleavage sites may require differing amounts of enzyme for complete cleavage. Thus the calculated amount of enzyme required to produce complete digestion of 1 µg of substrate DNA may not be accurate for a particular enzyme and substrate DNA. This difference is presumed to be due to an interaction between the enzyme and higher DNA structures around its recognition site. In fact, Nae I exhibits highly cleavage resistant sites (site preference) in pbr322 DNA. Experiments have shown that change in the composition of reaction mixture (i.e., addition of spermidine) may change the cleavage order or preference in such substrates. Reference 1. DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I Conrad, M. and Topal, M.D. (1989) Proc. Natl. Acad. Sci. USA 86: DNA Binding Substances When analyzing restriction digest results via gel electrophoresis, issues such as no visible bands, broad bands, and atypical migration of bands may be observed. These problems are generally caused by the DNA, the enzyme itself, or protein contaminants. Contaminating proteins can form protein-dna complexes which do not enter the gel or are not stained by ethidium bromide. Addition of denaturants such as SDS to the sample at a final concentration of around 0.1% will denature any complexes present and may improve gel results. Others Most restriction enzymes are guaranteed by the vendor to be stable for 1 2 years from the date of manufacture, However, this interval is somewhat arbitrary, and most enzymes do not lose their activity rapidly even after long-term storage (assuming use of the recommended storage conditions). Thus, there is a possibility that even enzymes stored for two years or longer can be active and still useful for digestion. For these enzymes, we recommend reassay before use, with experimental samples. Q&A The Relative Activity for each enzyme in the Universal Buffer supplied with the product is described in the Table on page 99. (Some restriction enzymes show higher activity in buffers other than the supplied universal buffer (for example, Hind III, whose supplied buffer is M, exhibits 200% of activity in buffer K). ) Q-1. Is there any reason not to use the higher-activity buffer for single digests? A-1. No. The supplied Universal Buffers are selected on the basis of similarity to the original basal buffer, and do not necessarily result in highest activity. However, we do not recommend use of buffers with bracketed numbers (see Table on page 99) with the selected enzyme, because they are prone to star activity. Q-2. Why does Takara supply a Universal Buffer for many of its restriction enzymes, when these enzymes exhibit lower activities in these buffers than that in the Basal Buffer? A-2. The Universal Buffers supplied with the enzyme are provided to facilitate convenient double digestions. Since the only difference between the L, M and H buffers is the salt concentration, a second digestion in a different buffer can be performed by simply adding the second enzyme and salt to a suitable concentration, rather than doing a buffer exchange or ethanol precipitation, etc. 2 Restriction Enzymes 97

99 2MOLECULAR BIOLOGY Restriction Enzymes TAKARA RESTRICTION ENZYMES A Basal Buffer is supplied with enzymes which do not exhibit sufficient activity in any of five of the Universal Buffers. Attention should be paid to the compositions of these buffers in double digests, since the Basal Buffers are supplied to provide optimal reaction conditions for each enzyme, and it may be difficult to do efficient double digestions without performing a complete buffer change (i.e., via dialysis or ethanol precipitation). Definition of Activity One unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete digest of 1 µg of l DNA in 60 minutes at 37 C in a 50 µl reaction volume. Substrate and temperature used in the activity determination are described for each enzyme. Relative activities of restriction enzyme activity using universal and basal buffers can be seen on page 208. In the relative activity table, buffers used in the activity determination and the recommended buffers are indicated for each enzyme. Residual Activity after Various Inactivating Procedures For each enzyme, residual activity was measured after the following 4 different inactivating procedures to examine the complete inactivation conditions. The results are summarized in the table on page Heating at 60 C for 15 minutes 2. Heating at 70 C for 15 minutes 3. Ethanol precipitation 4. Phenol treatment-ethanol precipitation Temperature Store each enzyme at 20 C. The enzyme solution does not freeze at 20 C. The enzyme will not be inactivated by one or two freeze-thaw cycles. However, repeated freeze-thaw cycles are not recommended. (An exception is Aat II, which should be stored at 80 C.) 10X Loading Buffer All Takara s restriction enzymes are supplied with 10X Loading Buffer (1 ml). Composition 1% SDS 50% Glycerol 0.05% Bromophenol Blue Restriction Enzyme-Universal Buffer Compatibility L M H K T+BSA Afa I Alu I Aor51H I Apa I ApaL I Ava II BspT104 I Bsp1286 I BssH II Dra I Eae I EcoO109 I Hae II Hae III Hap II Hha I Hinf I Kpn I Mbo II Mfl I Mlu I Msp I Nae I PmaC I Sac I Stu I Acc I Acc II Afa I Afl II* Alu I Aor51H I Ava I Ava II BspT104 I Bsp1407 I BssH II Bst1107 I Cla I Dra I Eae I EcoO109 I Eco81 I Fok I* EcoR V Hae II Hae III Hap II Hha I Hinc II Hind III Hinf I Hin1 I* Kpn I Mbo II Mfl I Mfu I Msp I Mun I* Nhe I PmaC I Psp1406 I Pvu II Sac I Sau3A I Sfi I Spe I Sse8387 I* Stu I Taq I Tth111 I Xba I* Xho I Xsp I Ban II Bgl II BmgT120 I BspT107 I BssH II BstP I BstX I Bst1107 I Cla I Cpo I Dra I EcoO65 I* EcoR I EcoR V EcoT14 I EcoT22 I Hae III Hha I Hinf I Mbo I Mlu I Mva I Nde I Not I** Pst I Sal I Sau3A I Sca I Smi I Spe I Sph I Stu I Taq I Van91 I VpaK11B I Xho I Afa I Aor13H I* BamH I Ban II Bcn I Bln I BmeT110 I BspT107 I Bst1107 I Cla I Cpo I Dra I EcoT14 I Fba I Hae II Hae III Hha I Hind III Hinf I Hpa I Mbo I Msp I Mva I Nco I* Nde I PshA I Pst I Nsb I Pvu I* Spe I Sph I Stu I Taq I Tth111 I Van91 I Xho I Xsp I Aat II Acc I Acc II Afa I Afl II Alu I Aor13H I Aor51H I ApaL I Ava I Ava II Bcn I BspT104 I Bsp1286 I Bsp1407 I BssH II Cla I Dra I Eae I EcoO109 I Eco81 I Hae II Hae III Hap II Hha I Hinc II Hinf I Hin1 I Mbo II Mfl I Mlu I Msp I Mun I Nae I Nsb I Nde I PmaC I Pdp1406 I PshA I Sac I Sac II Sfi I Sma I Stu I Taq I Xba I Xho I Xsp I * +0.01% BSA ** +0.01% BSA+0.01% Triton X

100 TAKARA RESTRICTION ENZYMES Legend for Restriction Enzymes Composition of Takara Universal Restriction Enzyme Buffers Buffer 10X L 100 mm Tris-HCl (ph 7.5) 100 mm MgCl 2 10 mm Dithiothreitol 10X M 100 mm Tris-HCl (ph 7.5) 100 mm MgCl 2 10 mm Dithiothreitol 500 mm NaCl 10X H 500 mm Tris-HCl (ph 7.5) 100 mm MgCl 2 10 mm Dithiothreitol 1,000 mm NaCl 10X K 200 mm Tris-HCl (ph 8.5) 100 mm MgCl 2 10 mm Dithiothreitol 1,000 mm KCl 10X T* 330 mm Tris-acetate (ph 7.9) 100 mm Magnesium acetate 5 mm Dithiothreitol 660 mm Potassium acetate * BSA or Triton X-100 should be added to a final concentration of 0.01% to obtain 100% activity. 10X Loading Buffer 1% SDS 0.05% Bromophenol blue 50% Glycerol CG CG dam dam dcm dcm Genomic DNA Analysis pkf3 Cloning Test Star Activity Reaction Temperature Reaction Temperature Reaction Temperature Reaction Temperature Reaction Temperature Reaction Temperature Enzyme activity is affected by CG methylase. Enzyme activity is not affected by CG methylase. Enzyme activity is affected by Dam methylase. Enzyme activity is not affected by Dam methylase. Enzyme activity is affected by Dcm methylase. Enzyme activity is not affected by Dcm methylase. CH + Methylation L M M + H H + Supplied Buffer= H + BSA H ++ Supplied Buffer= H + BSA + Triton X-100 Supplied Buffer= T T T + K K + B Ad2 pbr psw Supplied Buffer= L Supplied Buffer= M Supplied Buffer= M + BSA Supplied Buffer= H Supplied Buffer= T + BSA Supplied Buffer= K Supplied Buffer= K + BSA Supplied Buffer= Basal Substrate for unit definition: l DNA Substrate for unit definition: N 6 -methyladenine free l DNA Substrate for unit definition: Ad2 DNA Substrate for unit definition: pbr322 DNA Substrate for unit definition: pswa I - Eco O109 I DNA MOLECULAR BIOLOGY 2 Restriction Enzymes 99

101 2MOLECULAR BIOLOGY pkf3 Cloning Test pkf3 cloning test Quality control of restriction enzymes Takara follows a thorough QC process when manufacturing restriction enzymes. Takara performs several RNase, Protease, and DNase tests, as well as activity and nicking tests. Takara performs four additional quality tests. These tests include the Overdigestion test and Ligation-Recutting Test. Some enzymes are also analyzed by the pkf3 Cloning Test and the Genomic DNA Analysis. Table 1 Restriction enzymes tested using the pkf3 cloning test Acc III Aor13H I Aor51H I Ava II Bal I BamH I Ban II Bgl II BspT104 I BstP I Bst1107 I Cla I EcoO65 I EcoR I EcoT14 I Eco52 I Fba I Hind III Hpa I Kpn I Mlu I Nco I Nde I Not I Nsb I PshB I Pst I Pvu I Pvu II Sac I Sac II Sal I Sma I Sph I Sse8387 I Stu I VpaK11B I Xba I Xho I Restriction Enzymes Takara s Quality Control Protocol pkf3 cloning test 1. pkf3 DNA is digested using a 10-fold excess of a restriction enzyme having the unique site in multi-cloning site. 2. Following enzyme inactivation treatment, a portion of the digested DNA is ligated at 16 C for 30 minutes using DNA Ligation Kit, Ver. 1. (Cat.# 6021) 3. TH2 Competent Cells are transformed with part of this reaction mix, and cultured over two nights at 37 C on selective plates, LB-Cm-Sm plate (containing chloramphenicol and streptomycin), and LB-Cm plate (containing chloramphenicol). When exonuclease, etc., are absent from the restriction enzyme, colonies are not formed on LB-Cm-Sm plate since the transformant obtained is streptomycin sensitive. When restriction enzyme is contaminated with exonuclease, colonies form on LB-Cm-Sm medium as rpsl gene-deficient strain is obtained. The presence or absence of trace amounts of exonuclease can be judged depending on the presence or absence of colonies appearing on LB-Cm-Sm plate. Takara has confirmed that the ratio of rpsl gene-deficient transformant (number of colonies appearing on LB-Cm-Sm plate) against transformant (number of colonies appearing on LB-Cm plate) is less than 2% for all manufacturing lots of restriction enzymes. Figure 1. The Principle of pkf3 Cloning Test Culture Composition LB-Cm-Sm plate Yeast Extract... 5 mg/ml Peptone mg/ml NaCl... 5 mg/ml Chloramphenicol μg/ml Streptomycin μg/ml Agar % LB-Cm plate Yeast Extract... 5 mg/ml Peptone mg/ml NaCl... 5 mg/ml Chloramphenicol μg/ml Agar % 100

102 Restriction enzyme activity using universal and basal buffers Takara applies a system in which enzyme activities measured in the optimal universal buffers (one of 5 buffers: ) are displayed. The relative activity in other universal buffers is expressed as 100% of the optimal universal buffers. ( ) indicates buffers which are likely to invoke star activity. In order to avoid these effects, use of or buffers is recommended. For 15 enzymes (Acc III, Bal I, Bbe I, Bcn I, Bgl I, Bpu1102 I, Cfr10 I, Eam1105 I, Eco52 I, Nru I, PshB I, SnaB I, Ssp I, Taq I or VpaK11B I), basal buffer specialized for each enzyme is supplied. The compositions of basal buffers vary depending on enzymes. For double digestion reactions, recommended enzymes for each buffer (relative activity for each reaction mixture and enzyme) are listed. MOLECULAR BIOLOGY Relative activity for each reaction buffer supplied, activity measured buffer recommended buffer Restriction Relative Activities(%) Enzyme L M H K T+BSA Basal*** Aat II <20 <20 <20 < Acc I <20 (<20) Acc II (260) 100 < Acc III (<20) (<20) 20 (80) (<20) 100 Afa I Afl II 20 80* <20 < Alu I < Aor 13H I <20 20 <20 80* Aor 51H I < Apa I 100 <20 <20 <20 < Apa L I <20 < Ava II < Bal I <20 < BamH I (<20) < (<20) 80 Ban II (120) (120) (100) 100 Bcn I < Bgl I <20 < < Bgl II < (100) (60) 100 Bln I < BmeT110 I <20 < < BmgT120 I <20 < < Bpu1102 I <20 <20 < BspT104 I <20 < BspT107 I < Bsp1286 I <20 < Bsp1407 I BssH II Bst P I (<20) (60) 100 (100) (100) 100 Bst X I < <20 < Bst 1107 I (<20) Cfr 10 I (<20) (<20) (<20) 40 (20) 100 Cla I Cpo I <20 < < Dra I Dpn I Eae I <20 < Eam1105 I (<20) (40) (40) 100 EcoO65 I (20) (60) 60* EcoO109 I <20 < EcoR I (20) (100) 100 (120) (80) 120 EcoR V (<20) (40) 100 (120) (40) 100 EcoT14 I (<20) (40) (60) 100 EcoT22 I < (140) (20) 120 Eco52 I <20 <20 <20 <20 < Eco81 I < <20 < Fba I (<20) (<20) (80) 100 (20) 100 Fok I (20) (60)* <20 <20 (200) 100 Hae II < Hae III Restriction Enzyme Relative Activities(%) L M H K T+BSA Basal*** Hap II <20 < Hha I Hinc II Hind III (60) 100 < (100) 80 Hinf I Hin1 I 40 80* < Hpa I <20 (40) (80) 100 Kpn I <20 <20 (100) 80 Mbo I Mbo II <20 < Mfl I <20 < Mlu I (100) Msp I < Mun I (200) 100* <20 < Mva I (<20) (40) (20) 120 Nae I 100 <20 <20 < Nco I (40) (60) 20 60* (60) 160 Nde I < Nhe I (120) 100 <20 <20 (160) 100 Not I (<20) (<20) 20** <20 (<20) 100 Nru I 0 < < Nsb I < PmaC I <20 < PshA I < PshB I (20) (40) Psp1406 I <20 < Pst I (<20) (60) (20) 80 Pvu I (<20) (20) (40) 80* (40) 120 Pvu II (80) <20 (40) 100 Sac I <20 < Sac II <20 < Sal I <20 < (20) < Sau 3A I (60) <20 (80) 100 Sca I (<20) (<20) 100 (60) (<20) 100 Sfi I (40) 100 <20 < Sma I <20 <20 <20 < Smi I <10 < < SnaB I (20) (40) <20 <20 (40) 100 Spe I (80) (80) 100 Sph I (20) (40) (20) 100 Sse8387 I (120) 60* <20 <20 (60) 100 Ssp I (<20) (60) 40 (100) (80) 100 Stu I Taq I Tth111 I (20) (80) 120 Van91 I <20 (20) (60) 100 VpaK11B I <20 <20 60 (40) < Xba I <20 80* 20 < Xho I < Xsp I <20 60 < * +0.01% BSA 100% Afl II, Aor13H I, EcoO65 I, Fok I, Hin1 I, Mun I, Nco I, Pvu I, Sse8387 I, Xba I ** +0.01% BSA+0.01% Triton X % Not I *** The composition of basal buffers varies depending on enzymes. 2 Restriction Enzymes 101

103 Recommended Buffers for Double Digests 2MOLECULAR BIOLOGY Takara provides optimized buffers with each restriction enzyme and has determined the relative activity of each enzyme in these buffers (see page 99. In some situations, it may be necessary to perform a digestion with more than one restriction enzyme simultaneously or sequentially. This table provides information on the optimal buffer for a double digest reaction. The recommended buffer is shown for each combination of enzymes commonly used in cloning procedures. Some enzymes will function optimally at buffer concentrations other than 1X; these concentrations can be achieved by dilution or by adding more buffer as indicated. Restriction Enzymes Note: 1. It is confirmed that 10 units of each enzyme completley digests 1 µg of DNA at 37 C in one hour in 50 µl reaction mixture. 2. The concentration of glycerol should be less than 10% to minimize star activity. 3. DNA may not be digested completely when recognition sequences of 2 enzymes are close to each other, or when DNA forms a high structure combination

104 Fast Cutting Restriction Enzymes Rapid DNA restriction. Less waiting. Same price. MOLECULAR BIOLOGY Why wait for your restriction digestion reactions? Use just 1 μl of these Takara Bio restriction enzymes (RE) for complete, specific digestion of 1 μg plasmid in just 5 minutes with standard buffer and temperature conditions. Get research results faster with high-quality, rapid-cutting enzymes. Product Name Cat. No. Pack Size Cat. No. Pack Size Aat II 1112A 100 U 1112B 5x 100 U Acc I 1001A 100 U 1001B 5x 100 U Afa I (Rsa I) 1116A 1000 U 1116B 5x 1000 U Alu I 1004A 500 U 1004B 5x 500 U Apa I 1005A U 1005B 5x U Apa I (HC) 1005AH U 1005BH 5x U BamH I 1010A U 1010B 5x U BamH I (HC) 1010AH U 1010BH 5x U Ban II (HgiJ II) 1012A 2000 U 1012B 5x 2000 U BciT130 I (EcoR II, 1232A 2000 U 1232B 5x 2000 U Mva I) Bgl I 1020A 1000 U 1020B 5x 1000 U Bgl II 1021A 2000 U 1021B 5x 2000 U Bgl II (HC) 1021AH 2000 U 1021BH 5x 2000 U Bln I (Avr II) 1022A 400 U 1022B 5x 400 U BmeT110 I (Ava I) 1207A 500 U 1207B 5x 500 U BmgT120 I (Cfr13 I, 1231A 1000 U 1231B 5x 1000 U Asu I) Bsp1286 I (Sdu I) 1024A 500 U 1024B 5x 500 U BspT104 (Asu II, 1225A 2500 U 1225B 5x 2500 U Nsp V) BssH II (BseP I) 1119A 300 U 1119B 5x 300 U BstP I (BstE II) 1025A 2000 U 1025B 5x 2000 U BstX I 1027A 1000 U 1027B 5x 1000 U Cla I 1034A 1000 U 1034B 5x 1000 U Cla I (HC) 1034AH 1000 U 1034BH 5x 1000 U Cpo I (Rsr II) (Csp I) 1035A 400 U 1035B 5x 400 U Dra I (Aha III) 1037A 4000 U 1037B 5x 4000 U Dra I (Aha III) (HC) 1037AH 4000 U 1037BH 5x 4000 U Eae I (Cfr I) 1123A 200 U 1123B 5x 200 U Eco O65 I (BstE II) 1135A 1000 U 1135B 5x 1000 U EcoR I 1040A U 1040B 5x U EcoR I (HC) 1040AH U 1040BH 5x U EcoR V 1042A 3000 U 1042B 5x 3000 U EcoR V (HC) 1042AH 3000 U 1042BH 5x 3000 U EcoT22 I (Ava III) 1125A 2000 U 1125B 5x 2000 U (NsiI) Fok I 1046A 1000 U 1046B 5x 1000 U Hae III 1051A 4000 U 1051B 5x 4000 U Hae III (HC) 1051AH 4000 U 1051BH 5x 4000 U These enzymes are available in the Takara Starter Box (cat. no. 1400). Product Name Cat. No. Pack Size Cat. No. Pack Size Hap II (Hpa II) 1053A 2000 U 1053B 5x 2000 U Hap II (Hpa II) (HC) 1053AH 2000 U 1053BH 5x 2000 U Hha I 1056A 2000 U 1056B 5x 2000 U Hinf I 1061A 3000 U 1061B 5x 3000 U Hinf I (HC) 1061AH 3000 U 1061BH 5x 3000 U Hpa I 1064A 500 U 1064B 5x 500 U Mbo II 1145A 400 U 1145B 5x 400 U Nae I 1155A 500 U 1155B 5x 500 U Nco I 1160A 500 U 1160B 5x 500 U Nco I (HC) 1160BH 2500 U Not I 1166A 500 U 1166B 5x 500 U Not I (HC) 1166BH 2500 U Nru I 1168A 1000 U 1168B 5x 1000 U PshA I 1074A 200 U 1074B 5x 200 U PshB I (Vsp I) 1109A 1000 U 1109B 5x 1000 U Pst I 1073A U 1073B 5x U Pst I (HC) 1073AH U 1073BH 5x U Pvu II 1076A 2000 U 1076B 5x 2000 U Pvu II (HC) 1076AH 2000 U 1076BH 5x 2000 U Sac II 1079A 1000 U 1079B 5x 1000 U Sau3A I (Mbo I) 1082A 200 U 1082B 5x 200 U Sau3A I (Mbo I) (HC) 1082BH 1000 U Sca I 1084A 1500 U 1084B 5x 1500 U Sfi I 1178A 500 U 1178B 5x 500 U Sma I 1085A 2000 U 1085B 5x 2000 U Sma I (HC) 1085AH 2000 U 1085BH 5x 2000 U SnaB I 1179A 150 U 1179B 5x 150 U Spe I 1086A 300 U 1086B 5x 300 U Sph I 1180A 400 U 1180B 5x 400 U Sse8387 I 1183A 400 U 1183B 5x 400 U Sse8387 I (HC) 1183BH 2000 U Stu I 1088A 500 U 1088B 5x 500 U VpaK11B I (AvaII) 1196A 300 U 1196B 5x 300 U Xba I 1093A 3000 U 1093B 5x 3000 U Xba I (HC) 1093AH 3000 U 1093BH 5x 3000 U XspI (Bfa I, MaeI) 1095A 500 U 1095B 5x 500 U 2 Restriction Enzymes 103

105 MOLECULAR BIOLOGY Restriction Enzymes 2 T+ CG Aat II 1112A 1112B (A 5) Source: Genome DNA analysis: G ACGT C C TGCA G 100 units (4 12 units/µl) 500 units (4 12 units/µl) Acetobacter aceti Staphylococcus aureus genomic DNA M CG Acc I 1001A 1001B (A 5) Source: Genomic DNA analysis: 100 units (4 12 units/µl) 500 units (4 12 units/µl) Acinetobacter calcoaceticus Staphylococcus aureus genomic DNA Acc II (FnuD II) CG 1002A 1002B (A 5) Source: 100 units (4 12 units/µl) 500 units (4 12 units/µl) Acinetobacter calcoaceticus M CG CG GC GC B CG dam Acc III (BspM II) GT (A/C)(G/T) AC CA (T/G)(C/A) TG T CCGG A A GGCC T 1113A 20 units (1 5 units/µl) 1113B (A 5) 100 units (1 5 units/µl) Source: Acinetobacter calcoaceticus Effect of DNA methylation: This enzyme is capable of cleaving TC5mCGGA, but its rate is about 1/75 of cleavage efficiency in absence of methylation. Otherwise, enzyme activity is affected by Dam methylase depending on the sequence following the recognition site. Cleavage of pbr322: Commercially available pbr322 is substantially undigested, because the adenine residues in the recognition site (TCCGGA) are methylated by Dam methylase. CG CG Afa I (Rsa I) 1116A 1116B (A 5) Source: T+ GT AC CA TG 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Acidiphilium facilis M+ Afl II C TTAA G G AATT C 1003A 100 units (4 12 units/µl) 1003B (A 5) 500 units (4 12 units/µl) Source: Anabaena flos-aquae Ligation-Recutting Test: The ligation efficiency of cohesive ended DNA fragments generated by this enzyme will be lower than expected. More efficient ligation can be achieved using blunt end ligation reaction conditions. L Alu I 1004A 1004B (A 5) Source: Aor 13H I (BspM II, Acc III) 1224A 1224B (A 5) Source: enomic DNA G Analysis 104 AG CT TC GA 500 units (4 12 units/µl) 2500 units (4 12 units/µl) Arthrobacter luteus CG K+ T CCGG A A GGCC T 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Acidiphilium organovorum 13H Star Activity KF3 Cloning p Test CG E ffect of DNA Methylation Reaction Temperature S ubstrate for Unit Definition L Buffer

106 Aor51H I (Eco47 III) CG M AGC GCT 1118A 400 units (4 12 units/µl) TCG CGA 1118B (A 5) 2000 units (4 12 units/µl) Source: Acidiphilium organovorum 51H Genome DNA analysis: Staphylococcus aureus genomic DNA Apa I CG dcm L G GGCC C 1005A 10,000 units (8 20 units/µl) C CCGG G 1005AH 1005B (A 5) 1005BH (AH 5) 10,000 units (30 60 units/µl) 50,000 units (8 20 units/µl) 50,000 units (30 60 units/µl) Source: Acetobacter pasteurianus sub. pasteurianus Definition of activity: 3 units for l DNA as substrate is equivalent to 1 unit for an Ad2 substrate. MOLECULAR BIOLOGY ApaL I CG L G TGCA C 1006A 400 units (4 12 units/µl) C ACGT G 1006B (A 5) 2000 units (4 12 units/µl) Source: Acetobacter pasteurianus 2 Bal I 1009A 1009B (A 5) Source: Cleavage of pbr322: BamH I 1010A 1010AH (High concentration) 1010B (A 5) 1010BH (AH 5) (High conc.) Source: Notice: dcm B TGG CCA 20 units (1 5 units/µl) ACC GGT 100 units (1 5 units/µl) Brevibacterium albidum Commercially available pbr322 is substantially undigested, because the cytosine residues in the recognition site (TGGCCA) are methylated by Dcm methylase. CG dam dcm 10,000 units (8 20 units/µl) 10,000 units (30 60 units/µl) 50,000 units (8 20 units/µl) 50,000 units (30 60 units/µl) Bacillus amyloliquefaciens H This enzyme shows equivalent activity at 37 C and to 30 C, but the stability of the enzyme is lower in reaction mixtures at 37 C than that at 30 C. K G GATC C C CTAG G Restriction Enzymes Ban II (HgiJ II) CG H G (A/G)GC(T/C)C 1012A 2000 units (4 12 units/µl) C (T/C)CG(A/G)G 1012B (A 5) 10,000 units (4 12 units/µl) Source: Bacillus aneurinolyticus 105

107 B MOLECULAR BIOLOGY Bcn I (Cau II) 2 Bci T 130 I (EcoR II, Mva I) K dcm CC (A/T) GG GG (T/A) CC 1232A 2000 units (4 12 units/µl) 1232B (A 5) 10,000 units (4 12 units/µl) Source: Bacillus circulans T130 Ligation-recutting test: After digestion with this enzyme, the ligation is inefficient under the influence of the structure of Bci T130 I fragment ends. For ligation, the overnight ligation using DNA Ligation Kit Ver.2.1 is recommended. B CG Bgl I 1020A 1020B (A 5) Source: Genomic DNA analysis: GCCN NNN NGGC CGGN NNN NCCG 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Bacillus globigii Staphylococcus aureus genomic DNA H dam Bgl II Restriction Enzymes CC (C/G) GG GG (G/C) CC 1019A 1000 units (4 12 units/µl) 1019B (A 5) 5000 units (4 12 units/µl) Source: Bacillus centrosporus RFL 1 Ligation-recutting test: After digestion with this enzyme, inefficient ligations often are observed because of the structure of Bcn I fragment ends (Janulaitis, A.A., personal communication). For circularized ligation, overnight ligation using TaKaRa s DNA Ligation Kit, Ver. 2.1 or Mighty Mix Ligation Kit is recommended. 1021A 1021AH (High concentration) 1021B (A 5) 1021BH (AH 5) (High conc.) Source: A GATC T T CTAG A 2000 units (4 12 units/µl) 2000 units (30 60 units/µl) 10,000 units (4 12 units/µl) 10,000 units (30 60 units/µl) Bacillus globigii K Bln I (Avr II) C CTAG G G GATC C 1022A 400 units (4 12 units/µl) 1022B (A 5) 2000 units (4 12 units/µl) Source: Brevibacterium linens Genome DNA analysis: Escherichia coli genomic DNA Ligation-recutting test: The ligation efficiency of cohesive ends DNA fragments generated by this enzyme will be low. More efficient ligation can be achieved using the reaction conditions for blunt end ligation. K BmeT110 I (Ava I) C (T/C)CG(A/G) G G (A/G)GC(T/C) C 1207A 1207B (A 5) Source: 500 units (4 12 units/µl) 2500 units (4 12 units/µl) Bacillus megaterium T110 BmgT120 I (Cfr13 I, Asu I) CG dcm 1231A 1231B (A 5) Source: Effect of DNA methylation: 1,000 units (4 12 units/µl) 5,000 units (4 12 units/µl) Bacillus megalosporus T120 Enzyme activity is often affected by both Dcm and CG methylase. H G GNC C C CNG G B Bpu1102 I (Esp I) GC TNA GC CG ANT CG 1023A 150 units (4 12 units/µl) 1023B A 5) 750 units (4 12 units/µl) Source: Bacillus pumilus Ligation-recutting test: The ligation efficiency of the cohesive ended DNA fragments generated by this enzyme will be low. More efficient ligation can be achieved using blunt end ligation reaction conditions. enomic DNA G Analysis 106 Star Activity KF3 Cloning p Test CG E ffect of DNA Methylation Reaction Temperature S ubstrate for Unit Definition L Buffer

108 TT CG AA AA GC TT 2500 units (4 12 units/µl) 12,500 units (4 12 units/µl) Bacillus species T104 Arthrobacter luteus genomic DNA K BspT107 I (HgiC I) G G(T/C)(A/G)C C C C(A/G)(T/C)G G 1223A 1223B (A 5) Source: 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Bacillus species T107 Bsp1286 I (Sdu I) CG 1024A 1024B (A 5) Source: Notice: 500 units (4 12 units/µl) 2500 units (4 12 units/µl) Bacillus sphaericus Do not dilute. This enzyme is unstable at low concentrations. L T+ Bsp1407 I 1107A 1107B (A 5) Source: H CG G GTNAC C C CANTG G 2000 units (4 12 units/µl) 10,000 units (4 12 units/µl) Bacillus stearothermophilus H BstX I 1027A 1027B (A 5) Source: G CGCG C C GCGC G 300 units (4 12 units/µl) 1500 units (4 12 units/µl) Bacillus stearothermophilus H3 Staphylococcus aureus genomic DNA BstP I (BstE II, Eco065 I) 1025A 1025B (A 5) Source: CCAN NNNN NTGG GGTN NNNN NACC 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Bacillus stearothermophilus K CG Bst1107 I (Sna I) 1028A 1028B (A 5) Source: Genome DNA analysis: 150 units (4 12 units/µl) 750 units (4 12 units/µl) Bacillus stearothermophilus RFL 1107 Arthrobacter luteus genomic DNA Cfr10 I CG 1120A 1120B (A 5) Source: 200 units (4 12 units/µl) 1000 units (4 12 units/µl) Citrobacter freundii RFL 10 2 B GTA TAC CAT ATG (A/G) CCGG (T/C) (T/C) GGCC (A/G) 107 Restriction Enzymes 1119A 1119B (A 5) Source: Genome DNA analysis: M CG BssH II (BseP I) G(A/G/T)GC(A/C/T) C C (T/C/A)CG(T/G/A)G T GTAC A A CATG T 300 units (4 12 units/µl) 1500 units (4 12 units/µl) Bacillus stearothermophilus RFL 1407 MOLECULAR BIOLOGY 1225A 1225B (A 5) Source: Genome DNA analysis: L CG BspT104 I (Asu II, Nsp V)

109 MOLECULAR BIOLOGY Restriction Enzymes 2 M CG dam Cla I 1034S 1034A 1034AH (high conc.) 1034B (A 5) 1034BH (AH 5) (high conc.) Source: Genome DNA analysis: Notice: AT CG AT TA GC TA 500 units (4 12 units/µl) 1000 units (4 12 units/µl) 1000 units (30 60 units/µl) 5000 units (4 12 units/µl) 5000 units (30 60 units/µl) Caryophanon latum L Arthrobacter luteus genomic DNA This enzyme shows equivalent activity at 37 C and 30 C. K CG Cpo I (Rsr II) 1035A 400 units (4 12 units/µl) 1035B (A 5) 2000 units (4 12 units/µl) Source: Caseobacter polymorphus Genome DNA analysis: Staphylococcus aureus genomic DNA Notice: Do not dilute. This enzyme is unstable at low concentration. The relative activity of this enzyme is 50% at 37 C, and 120% at 25 C. Addition of BSA to a final concentration of 0.01% in the reaction mixture increases the relative activity up to 120% at 30 C and up to 160% at 25 C. CG G(A/T)C CG GC C(T/A)G GC M Dra I (Aha III) 1037A 1037AH (high conc.) 1037B (A 5) 1037BH (AH 5) (high conc.) Source: Genome DNA analysis: TTT AAA AAA TTT 4000 units (8 20 units/µl) 4000 units (30 60 units/µl) 20,000 units (8 20 units/µl) 20,000 units (30 60 units/µl) Deinococcus radiophilus Pseudomonas aeruginosa genomic DNA T dam CG Dpn I G MA T C C T AMG 1235A 1000 units (10 units/µl) 1235B 5000 units (10 units/µl) Source: Escherichia coli Notice: This enzyme can cut the sequence GmATC (A is methylated) and cannot cut the sequence GATC (A is not methylated). This enzyme can cut DNA prepared from commonly used E. coli strains (Dam+ strain), but not PCR products. Enzyme activity is not affected by Dam methylase. M Eae I (Cfr I) CG dcm 1123A 1123B (A 5) Source: 200 units (4 12 units/µl) 1000 units (4 12 units/µl) Enterobacter aerogenes CG Eam1105 I (T/C) GGCC (A/G) (A/G) CCGG (T/C) B 1124A 100 units (2 10 units/µl) 1124B (A 5) 500 units (2 10 units/µl) Source: Enterobacter amnigenes RFL 1105 Ligation-recutting test: Ligation efficiency of cohesive ended DNA fragments generated by this enzyme is low. More efficient ligation can be achieved using blunt end ligation reaction conditions. Eco065 I (BstE II, BstP I) 1135A 1135B (A 5) Source: Star activity: enomic DNA G Analysis 108 CG GACNN N NNGTC CTGNN N NNCAG H+ G GTNAC C C CANTG G 1,000 units (4 12 units/µl) 5,000 units (4 12 units/µl) Escherichia coli K11a Nonspecific cleavage often occurs. Star Activity KF3 Cloning p Test CG E ffect of DNA Methylation Reaction Temperature S ubstrate for Unit Definition L Buffer

110 L dcm 1043A 2,000 units (8 20 units/µl) 1043B (A 5) 10,000 units (8 20 units/µl) Source: Escherichia coli H709C Effect of DNA methylation: Enzyme activity is affected by Dcm methylase, depending on the sequence following the recognition site. 1040A 1040AH (high conc) 1040B (A 5) 1040BH (AH 5) (high conc.) Source: H CG EcoR I 10,000 units (8 20 units/µl) 10,000 units (30 60 units/µl) 50,000 units (8 20 units/µl) 50,000 units (30 60 units/µl) Escherichia coli RY 13 H EcoR V CG 1042A 1042AH (high conc.) 1042B (A 5) 1042BH (AH 5) (high conc.) Source: 3000 units (8 20 units/µl) 3000 units (30 60 units/µl) 15,000 units (8 20 units/µl) 15,000 units (30 60 units/µl) Escherichia coli H EcoT14 I (Sty I) 1038A 1038B (A 5) Source: 3000 units (4 12 units/µl) 15,000 units (4 12 units/µl) Escherichia coli TB units (4 12 units/µl) 10,000 units (4 12 units/µl) Escherichia coli WA A 1039B (A 5) Source: Genome DNA analysis: Effect of DNA methylation: B CG Eco52 I (Xma III) 200 units (4 12 units/µl) 1,000 units (4 12 units/µl) Escherichia coli RFL 52 Staphylococcus aureus genomic DNA Enzyme activity is affected by CG methylase. M Eco81 I (Sau I) 1131A 500 units (4 12 units/µl) 1131 (A 5) 2,500 units (4 12 units/µl) Source: Escherichia coli RFL 8 Ligation-recutting test: After digestion with this enzyme, ligation is inefficient due to the structure of the Eco81 I fragment ends (Janulaitis, A.A., personal communication). For circularized ligation, the overnight ligation using TaKaRa DNA Ligation Kit, Ver. 1 or Ver. 2.1 or Mighty Mix is recommended. K dam Fba I (Bcl I) 1045A 500 units (4 12 units/µl) 1045B (A 5) 2,500 units (4 12 units/µl) Source: Flavobacterium balustinum Effect of DNA methylation: Enzyme activity is affected by Dam methylase. Therefore, most DNAs from E. coli are not cleavable. M+ Fok I CG 1046A 1046B (A 5) Source: Effect of DNA methylation: 1,000 units (4 12 units/µl) 5,000 units (4 12 units/µl) Escherichia coli UT 481 carrying plasmid encoding Fok I gene Enzyme activity is not affected by CG methylase. G AATT C C TTAA G GAT ATC CTA TAG C C(A/T)(A/T)G G G G(T/A)(T/A)C C A TGCA T T ACGT A C GGCC G G CCGG C CC TNA GG GG ANT CC T GATC A A CTAG T GGATGNNNNNNNNN CCTACNNNNNNNNN NNNN Restriction Enzymes H EcoT22 I (Ava III) 1125A 1125B (A 5) Source: (A/G)G GNC C(T/C) (T/C)C CNG G(A/G) MOLECULAR BIOLOGY Eco0109 I (Dra II)

111 2MOLECULAR BIOLOGY Hae II CG M (A/G) GCGC (T/C) 1052A 1,000 units (4 12 units/µl) (T/C) CGCG (A/G) 1052B (A 5) Source: 5,000 units (4 12 units/µl) Haemophilus aegyptius Hae III CG M GG CC 1051A 4,000 units (4 12 units/µl) CC GG 1051AH (High conc.) 1051B (A 5) 1051BH (AH 5) (High conc.) Source: 4,000 units (30 60 units/µl) 20,000 units (4 12 units/µl) 20,000 units (30 60 units/µl) Haemophilus aegyptius Hap II (Hpa II, Msp I) CG L C CG G 1053A 2,000 units (4 12 units/µl) G GC C 1053AH (High conc.) 1053B (A 5) 1053BH (AH 5) (High conc.) Source: 2,000 units (30 60 units/µl) 10,000 units 4 12 units/µl) 10,000 units (30 60 units/µl) Haemophilus aphrophilus Hha I CG G CG C 1056A 2,000 units (4 12 units/µl) C GC G 1056B (A 5) Source: Effect of DNA methylation: 10,000 units (4 12 units/µl) Haemophilus haemolyticus Enzyme activity is affected by CG methylase. Restriction Enzymes Hinc II (Hind II) CG M GT(T/C) (A/G)AC 1059A 1,000 units (4 12 units/µl) CA(A/G) (T/C)TG 1059AH (High conc.) 1059B (A 5) 1059BH (AH 5) (High conc.) Source: Effect of DNA methylation: 1,000 units (30 60 units/µl) 5,000 units (4 12 units/µl) 5,000 units (30 60 units/µl) Haemophilus influenzae Rc Enzyme activity is not affected by CG methylase. Hind III M A AGCT T 1060A 10,000 units (8 20 units/µl) T TCGA A 1060AH (High conc.) 1060B (A 5) 1060BH (AH 5) (High conc.) Source: 10,000 units (30 60 units/µl) 50,000 units (8 20 units/µl) 50,000 units (30 60 units/µl) Haemophilus influenzae Rd Hinf I CG H G ANT C 1061A 3,000 units (4 12 units/µl) C TNA G 1061AH (High conc.) 1061B (A 5) 1061BH (AH 5) (High conc.) Source: Effect of DNA methylation: 3,000 units (30 60 units/µl) 15,000 units (4 12 units/µl) 15,000 units (30 60 units/µl) Haemophilus influenzae RF Enzyme activity is not affected by CG methylase. Genomic DNA Analysis Star Activity pkf3 Cloning Test CG Effect of DNA Methylation Reaction Temperature Substrate for Unit Definition L Buffer 110

112 Hin1 I (Acy I, Bbi II) CG M + G(A/G) CG (T/C)C 1057A 300 units (4 12 units/µl) C(T/C) GC (A/G)G 1057B (A 5) 1500 units (4 12 units/µl) Source: Haemophilus influenzae RFL 1 Hpa I K GTT AAC 1064A 500 units (4 12 units/µl) CAA TTG 1064B (A 5) Source: 2500 units (4 12 units/µl) Haemophilus parainfluenzae MOLECULAR BIOLOGY Kpn I CG L G GTAC C 1068A 5000 units (4 12 units/µl) C CATG G 1068AH (High conc.) 1068B (A 5) 1068BH (AH 5) (High conc.) Source: 5000 units (30 60 units/µl) 25,000 units (4 12 units/µl) 25,000 units (30 60 units/µl) Klebsiella pneumoniae Mbo I (Sau3A I) CG dam K GATC 1069A 1000 units (4 12 units/µl) CTAG 1069B (A 5) Source: Effect of DNA methylation: 5000 units (4 12 units/µl) Moraxella bovis Enzyme activity is affected by Dam methylase, but not by CG methylase. Most generally available DNAs from E. coli are not cleavable. 2 Mbo II CG dam L GAAGANNNNNNN N 1145A 400 units (4 12 units/µl) CTTCTNNNNNNN 1145B (A 5) Source: Ligation-recutting test: Effect of DNA methylation: Mfl I (Xho II) 1070A 1070B (A 5) Source: Effect of DNA methylation: 2000 units (4 12 units/µl) Moraxella bovis The ligation efficiency of cohesive ended DNA fragments with generated by this enzyme is low. More efficient ligation can be achieved using blunt end ligation reaction conditions. Enzyme activity is affected by Dam methylase, depending on the sequence following the recognition site. It is not affected by CG methylase. dam L (A/G) GATC (T/C) 500 units (4 12 units/µl) (T/C) CTAG (A/G) 2500 units (4 12 units/µl) Microbacterium flavum Enzyme activity is affected by Dam methylase. Therefore, generally available DNAs from E. coli are not cleavable. Restriction Enzymes Mlu I CG H A CGCG T 1071A 1000 units (4 12 units/µl) T GCGC A 1071AH (High conc.) 1071B (A 5) 1071BH (AH 5) (High conc.) Source: Genome DNA analysis: Effect of DNA methylation: 1000 units (30 60 units/µl) 5000 units (4 12 units/µl) 5000 units (30 60 units/µl) Micrococcus luteus Staphylococcus aureus genomic DNA Enzyme activity is affected by CG methylase. Msp I (Hpa II, Hap II) CG T + C CG G 1150A 3000 units (8 20 units/µl) G GC C 1150AH (High conc.) 1150B (A 5) 1150BH (AH 5) (High conc.) Source: Effect of DNA methylation: 3000 units (30 60 units/µl) 15,000 units (8 20 units/µl) 15,000 units (30 60 units/µl) Moraxella species Enzyme activity is not affected by CG methylase

113 2MOLECULAR BIOLOGY Mun I (Mfe I) M + C AATT G 1153A 150 units (4 12 units/µl) G TTAA C 1153B (A 5) Source: Genome DNA analysis: 750 units (4 12 units/µl) Escherichia coli carrying a plasmid encoding Mun I gene Arthrobacter luteus genomic DNA Nae I CG pbr L 1155A 1155B (A 5) Source: Genome DNA analysis: Effect of DNA methylation: Notice: 500 units (4 12 units/µl) 2,500 units (4 12 units/µl) Nocardia aerocolonigenes Staphylococcus aureus genomic DNA Enzyme activity is affected by CG methylase. This enzyme is able to cleave 4 pbr322 sites, but the site at position 1283 is resistant to complete cleavage. GCC GGC CGG CCG Nco I K + C CATG G 1160A 500 units (4 12 units/µl) G GTAC C 1160B (A 5) 1160BH (High conc.) Source: 2,500 units (4 12 units/µl) 2,500 units (30 60 units/µl) Nocardia carollina Restriction Enzymes Nde I H CA TA TG 1161A 400 units (4 12 units/µl) GT AT AC 1161B (A 5) Source: Ligation-recutting test: 2,000 units (4 12 units/µl) Neisseria denitrificans Ligation efficiency of the cohesive ended DNA fragments generated by this enzyme is low. More efficient ligation can be achieved using blunt end ligation reaction conditions. Notice: Do not dilute. This enzyme is unstable at low concentrations. The addition of Triton-X 100 to a final concentration of 0.01% in the reaction mixture increases the relative activity to approximately 150%. Nhe I CG M G CTAG C 1162A 500 units (4 12 units/µl) C GATC G 1162B (A 5) Source: Genomic DNA analysis: Effect of DNA methylation: 2,500 units (4 12 units/µl) Neisseria mucosa heidelbergensis Arthrobacter luteus genomic DNA Enzyme activity is affected by CG methylase depending on the sequence following the recognition site. Not I CG Ad2 H + GC GGCC GC 1166A 500 units (4 12 units/µl) CG CCGG CG 1166B (A 5) 1166BH (High conc.) Source: Substrate for unit definition: Genomic DNA analysis: Effect of DNA methylation: 2,500 units (4 12 units/µl) 2,500 units (30 60 units/µl) Nocardia otitidis-caviarum Ad2 DNA Escherichia coli genomic DNA Enzyme activity is affected by CG methylase. Genomic DNA Analysis Star Activity pkf3 Cloning Test CG Effect of DNA Methylation Reaction Temperature Substrate for Unit Definition L Buffer 112

114 TCG CGA AGC GCT 1000 units (4 12 units/µl) 5000 units (4 12 units/µl) Nocardia rubra Staphylococcus aureus genomic DNA T+ CG Nsb I (Mst I, Avi II, Fsp I) 1226A 1226B (A 5) Source: Genomic DNA analysis: 200 units (4 12 units/µl) 1,000 units (4 12 units/µl) Neisseria subtlava Va-1 Arthrobacter luteus chromosomal DNA PmaC I CG 1177A 1177B (A 5) Source: 500 units (4 12 units/µl) 2,500 units (4 12 units/µl) Pseudomonas maltophila CB50P PshA I CG TGC GCA ACG CGT L CAC GTG GTG CAC K B 1109A 1109B (A 5) Source: Genome DNA analysis: Psp1406 I (Acl I) 1108A 1108B (A 5) Source: Genomic DNA analysis: T+ Pvu I AA CG TT TT GC AA 200 units (4 12 units/µl) 1,000 units (4 12 units/µl) Pseudomonas species RFL1406 Arthrobacter luteus genomic DNA H Pst I 1073A 1073AH (High conc.) 1073B (A 5) 1073BH (AH 5) (High conc.) Source: AT TA AT TA AT TA 1,000 units (4 12 units/µl) 5,000 units (4 12 units/µl) Plesiomonas shigelloides Pseudomonas aeruginosa genomic DNA CG 10,000 units (8 20 units/µl) 10,000 units (30 60 units/µl) 50,000 units (8 20 units/µl) 50,000 units (30 60 units/µl) Escherichia coli ED8654 carrying the plasmid encoding Pst I gene CG dam GACNN NNGTC CTGNN NNCAG K+ 1075A 200 units (4 12 units/µl) 1075B (A 5) 1,000 units (4 12 units/µl) Source: Proteus vulgaris Genomic DNA analysis: Flavobacterium okeanokoites genomic DNA Ligation-recutting test: The ligation efficiency of cohesive ended DNA fragments generated by this enzyme will be low. More efficient ligation can be achieved using blunt end ligation reaction conditions. Effect of DNA methylation: Enzyme activity is affected by CG methylase, but not Dam methylase. C TGCA G G ACGT C CG AT CG GC TA GC Restriction Enzymes 1074A 200 units (4 12 units/µl) 1074B (A 5) 1,000 units (4 12 units/µl) Source: Plesiomonas shigelloides Effect of DNA methylation: Enzyme activity is affected by CG methylase depending on the sequence of the recognition site. Notice: Do not dilute. This enzyme is unstable at low concentrations. Addition of BSA to a final concentration of 0.01% in the reaction mixture increases its relative activity to 140% at 37 C and 220% at 30 C. PshB I (Vsp I) MOLECULAR BIOLOGY 1168A 1168B (A 5) Source: Genomic DNA analysis: B CG dam Nru I

115 M MOLECULAR BIOLOGY Pvu II A 1076AH (high conc.) 1076B (A 5) 1076BH (A 5) (high conc.) Source: L CG Sac I 1078A 1078AH (High conc.) 1078B (A 5) 1078BH (A 5) (High conc.) Source: Effect of DNA methylation: G AGCT C C TCGA G 2,000 units (4 12 units/µl) 2,000 units (30 60 units/µl) 10,000 units (4 12 units/µl) 10,000 units (30 60 units/µl) Streptomyces achromogenes Enzyme activity is not affected by CG methylase. Ad2 T+ CG Sac II CC GC GG GG CG CC 1079A 1,000 units (8 20 units/µl) 1079B (A 5) 5,000 units (8 20 units/µl) Source: Streptomyces achromogenes Genome DNA analysis: Staphylococcus aureus genomic DNA Effect of DNA methylation: Enzyme activity is affected by CG methylase. Notice: This enzyme is able to cleave at 4 l DNA sites, but site at position is resistant to cleavage. H CG Sal I Restriction Enzymes CAG CTG GTC GAC 2,000 units (8 20 units/µl) 2,000 units (30 60 units/µl) 10,000 units (8 20 units/µl) 10,000 units (30 60 units/µl) Proteus vulgaris 1080A 1080AH (High conc.) 1080B (A 5) 1080BH (AH 5) (High conc.) Source: Genome DNA analysis: Effect of DNA methylation: G TCGA C C AGCT G 3,000 units (8 20 units/µl) 3,000 units (30 60 units/µl) 15,000 units (8 20 units/µl) 15,000 units (30 60 units/µl) Streptomyces albus G Staphylococcus aureus genomic DNA Enzyme activity is affected by CG methylase. H CG dam Sau3A I (Mbo I) GATC CTAG 1082A 200 units (4 12 units/µl) 1082B (A 5) 1,000 units (4 12 units/µl) 1082BH (High conc.) 1,000 units (30 60 units/µl) Source: Staphylococcus aureus 3A Effect of DNA methylation: Enzyme activity is affected by CG methylase, depending on the sequence following the recognition site, but is not affected by Dam methylase. H Sca I AGT ACT TCA TGA 1084A 1,500 units (4 12 units/µl) 1084B (A 5) 7,500 units (4 12 units/µl) Source: Streptomyces caespitosus Star activity: Unrelated sites may often be cut in the presence of Mn2+, and at alkaline ph or low ionic strength. Notice: Do not dilute. This enzyme is unstable at low concentrations. Addition of BSA to a final concentration of 0.01% in reaction mixture increases the relative activity up to 120% at 30 C and up 240% at 25 C, but does not increase the activity at 37 C. dcm Sfi I 1178A 1178B (A 5) Source: Genomic DNA analysis: enomic DNA G Analysis 114 Ad2 M GGCCN NNN NGGCC CCGGN NNN NCCGG 500 units (4 12 units/µl) 2500 units (4 12 units/µl) Streptomyces fimbriatus Escherichia coli genomic DNA Star Activity KF3 Cloning p Test CG E ffect of DNA Methylation Reaction Temperature S ubstrate for Unit Definition L Buffer

116 1085A 2,000 units (4 12 units/µl) 1085AH (High conc.) 2,000 units (30 60 units/µl) 1085B (A 5) 10,000 units (4 12 units/µl) 1085BH (AH 5) (High conc.) 10,000 units (30 60 units/µl) Source: Serratia marcescens Sb Genome DNA analysis: Staphylococcus aureus genomic DNA Notice: This enzyme shows equivalent activity at 37 C and to 30 C, but the stability of the enzyme in reaction mixtures is lower at 37 C than at 30 C. Ad2 Smi I (Swa I) 1111A 1111B (A 5) Source: Genomic DNA analysis: 200 units (10 units/µl) 1,000 units (10 units/µl) Steptococcus milleris Escherichia coli genomic DNA 150 units (4 12 units/µl) 750 units (4 12 units/µl) Sphaerotilus natans Arthrobacter luteus genomic DNA Enzyme activity is affected by CG methylase. Ad2 M Spe I 300 units (4 12 units/µl) 1,500 units (4 12 units/µl) Sphaerotilus natans Escherichia coli genomic DNA 1180A 1180B (A 5) Source: Effect of DNA methylation: H CG Sph I 400 units (4 12 units/µl) 2,000 units (4 12 units/µl) Streptomyces phaeochromogenes Enzyme activity is affected by CG methylase. M+ Sse8387 I 1183A 1183B (A 5) 1183BH (High conc.) Source: Genomic DNA analysis: 400 units (4 12 units/µl) 2,000 units (4 12 units/µl) 2,000 units (30 60 units/µl) Streptomyces species Staphylococcus aureus genomic DNA B Ssp I 1185A 1185B (A 5) Source: Genomic DNA analysis: 500 units (4 12 units/µl) 2,500 units (4 12 units/µl) Sphaerotilus natans Arthrobacter luteus genomic DNA Stu I dcm M 1088A 500 units (4 12 units/µl) 1088B (A 5) 2,500 units (4 12 units/µl) Source: Streptomyces tubercidicces Effect of DNA methylation: Enzyme activity is affected by Dcm methylase, depending on the sequence following the recognition site. ATTT AAAT TAAA TTTA TAC GTA ATG CAT A CTAG T T GATC A G CATG C C GTAC G CC TGCA GG GG ACGT CC AAT ATT TTA TAA AGG CCT TCC GGA Restriction Enzymes 1086A 1086B (A 5) Source: Genomic DNA analysis: B CG SnaB I 1179A 1179B (A 5) Source: Genomic DNA analysis: Effect of DNA methylation: H CCC GGG GGG CCC MOLECULAR BIOLOGY T+ CG Sma I

117 MOLECULAR BIOLOGY Restriction Enzymes T CG A A GC T 1189A 2,000 units (4 12 units/µl) 1189B (A 5) 10,000 units (4 12 units/µl) Source: Thermus aquaticus YT-1 Effect of DNA methylation: Enzyme activity is affected by Dam methylase, depending on the sequence following the recognition site, but is not affected by CG methylase. K Tth111 I GACN N NGTC CTGN N NCAG 1090A 500 units (4 12 units/µl) 1090B (A 5) 2,500 units (4 12 units/µl) Source: Thermus thermophilus strain 111 Ligation-recutting test: The ligation efficiency cohesive ended DNA fragments with generated by this enzyme is low. More efficient ligation can be achieved using blunt end ligation reaction conditions. K dcm Van91 I (PflM I) 2 B CG dcm Taq I (TthHB8 I) 1193A 300 units (4 12 units/µl) 1193B (A 5) 1,500 units (4 12 units/µl) Source: Vibrio anguillarum RFL 91 Effect of DNA methylation: Enzyme activity is affected by Dcm methylase depending on the sequence of the recognition site. B CG dcm VpaK11B I (Ava II) CCAN NNN NTGG GGTN NNN NACC G G(A/T)C C C C(T/A)G G 1196A 300 units (4 12 units/µl) 1196B (A 5) 1,500 units (4 12 units/µl) Source: Vibrio parahaemolyticus Effect of DNA methylation: Enzyme activity is affected by Dcm methylase or CG methylase depending on the sequence following the recognition site. M+ dcm Xba I T CTAG A A GATC T 1093A 3,000 units (8 20 units/µl) 1093AH (High conc.) 3,000 units (30 60 units/µl) 1093B (A 5) 15,000 units (8 20 units/µl) 1093BH (AH 5) (High conc.) 15,000 units (30 60 units/µl) Source: Xanthomonas badrii Genome DNA analysis: Escherichia coli genomic DNA Effect of DNA methylation: Enzyme activity is affected by Dam methylase depending on the sequence following the recognition site. CG Xho I H C TCGA G G AGCT C 1094A 5000 units (4 12 units/µl) 1094AH (High conc.) 5000 units (30 60 units/µl) 1094B (A 5) 25,000 units (4 12 units/µl) 1094BH (AH 5) (High conc.) 25,000 units (30 60 units/µl) Source: Xanthomonas holcicola Genome DNA analysis: Escherichia coli genomic DNA Ligation-recutting test: The ligation efficiency of cohesive ends DNA fragments generated by this enzyme will be low. More efficient ligation can be achieved using reaction conditions for blunt end ligation. Effect of DNA methylation: Enzyme activity is affected by CG methylase. (It cleaves CT5m CGAG very slowly.) K Xsp I (Bfa I, Mae I) C TA G C AT C 1095A 500 units (4 12 units/µl) 1095B (A 5) 2500 units (4 12 units/µl) Source: Xanthomonas species YK1 Ligation-recutting test: The ligation efficiency of cohesive ends DNA fragments generated by this enzyme will be low. More efficient ligation can be achieved using reaction conditions for blunt end ligation. enomic DNA G Analysis 116 Star Activity KF3 Cloning p Test CG E ffect of DNA Methylation Reaction Temperature S ubstrate for Unit Definition L Buffer

118 IPTG (dioxane free) (Isopropyl-b-D-thiogalactopyranoside) IPTG (dioxane free) (Isopropyl-b-D-thiogalactopyranoside) Cat.# g An Inducer of b-galactosidase Expression in Bacteria Used with X-Gal (Cat.# 9031) for blue/white colony screening to distinguish non-recombinant and recombinant colonies An Inducer of the lac and tac Promoters Property Molecular weight: Purity Biological activity as an inducer of b-galactosidase activity inducer was examined Purity was examined by TLC analysis Form White powder 20 C MOLECULAR BIOLOGY X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-Galactoside) X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-Galactoside) Cat.# g A Histochemical Substrate for b-galactosidase and that Yields a Blue Precipitate upon Hydrolysis Used with X-Gal (Cat.# 9031) for blue/white colony screening to distinguish non-recombinant and recombinant colonies Property Molecular weight: Purity Biological activity as the substrate for b-galactosidase was examined Purity was examined by TLC analysis Form White powder 20 C 2 Others Proteinase K Proteinase K Cat.# ml Purification of DNA, RNA and Phage Preparation of Chromosomal DNA for in situ Hybridization and Fingerprinting, Colony Hybridization, Plaque Hybridization, Pulsed-Field Electrophoresis Proteinase K is a high activity serine protease. Its activity is stable in the presence of calcium salt, and increases in the presence of protein denaturants such as SDS or urea. Proteinase K can be used on a wide range of substrates. It degrades ester and peptide bonds preferentially next to C-termini of hydrophobic, sulfuric, or aromatic amino acids. Concentration 20 mg/ml Source Tritirachium album Form 20 mm Tris-HCl (ph7.4), 1 mm CaCl 2, 50% glycerol Purity Endonuclease, Exonuclease, Nickase and RNase activity were not detected as judged by gel electrophoresis Definition of Activity One unit of enzyme activity is defined as the amount required to liberate Folin positive amino acid corresponding to 1 µmol of tyrosine using urea-denatured bovine hemoglobin denatured by urea at 37 C for 1 minute. Activity $ 30 units/mg protein 20 C Enzyme Code

119 2MOLECULAR BIOLOGY Others Westase Enzyme Westase Enzyme Cat.# g Application Digestion of Yeast Cell Walls Westase Enzyme possesses lytic yeast cell walls. Its primary enzyme activities consist of b-1,6 glucanase and b-1,3 glucanase activity. This enzyme works well for ascosporogenous yeasts such as Saccharomyces cerevisiae. It can also be used with fission yeasts such as Schizosaccharomyces pombe, which cannot be protoplasted with Zymolyase treatment, or basidiosporogenous yeasts. * Manufactured by Ozeki Corporation. Source Streptomyces rochei DB-34 Westase Enzyme is prepared from liquid culture supernatant of Streptomyces rochei DB-34. Agarose L03 Agarose L03 is recommended for separations of DNA fragments $1,000 bp. Properties Gelling Temperature C EEO (Electroendosmosis) (-Mr) Gel strength (1.5%) $2,200 g/cm 2 Sulfate #0.09% Form Lyophilized powder (containing celite as the excipient) Definition of Activity b-1,6 glucanase activity: One unit is defined as the amount required to release 1 µmol reducing sugar from 10 mg/ml Pustulan solution in 1 min. at 37 C, ph 6.0. Lytic activity: One unit is defined as the amount required to cause a 1% decrease in absorbance at 660 nm in 1 min. at 30 C, ph 6.0 when using cell wall fraction of Cryptococcus albidus IFO 0612 as a substrate. Specific Activities b-1,6 glucanase activity: Lytic activity: DNase activity: 4 C (with dessicant) $ 400 units/g > 35,000 units/g ND Agarose LO3 Cat.# g Room temperature 118

120 Introduction Takara s Cell Biology and Antibody Products Enzyme Immunoassay kits Takara s EIA Kit Summary of Takara EIA Kits Bone Research Osteocalcin Products Porcine Pig Gla-Osteocalcin and Glu-Osteocalcin EIA Kits NEW. 123 Human Gla-Osteocalcin High Sensitive EIA Kit Mouse Gla-Osteocalcin High Sensitive EIA Kit NEW Multi-Species Gla-type Osteocalcin (Gla-OC) EIA Kit (Precoated) Undercarboxylated (Glu-OC) EIA Kit Rat Rat Gla/Glu-Osteocalcin High Sensitive EIA Kit NEW Other Bone Research Products Osteoblast-Inducer Reagent (for animal cell) TRACP & ALP Double Stain Kit TRACP & ALP Assay Kit Procollagen Type I C-Peptide (PIP) EIA Kit (Precoated) RetroNectin EIA Kit NEW Cell Adhesion & Extracellular Matrix Fibronectin Products Fibronectin EIA Kit (Precoated) Fibronectin-Related Peptides Laminin Products Laminin EIA Kit (Precoated) Laminin Peptides Vitronectin Products Vitronectin EIA Kit (Precoated) Heparan Degrading Enzyme EIA Kit Glycocalicin EIA Kit, Ver Human E-cadherin EIA Kit GMP-140 (P-selectin) EIA Kit Cell Differentiation Kit GPDH Activity Assay Kit Cell Proliferation & Viability Rat Heme Oxygenase-1 EIA KIt Mouse Heme Oxygenase-1 EIA Kit (Precoated) Signal Transduction Universal Tyrosine Kinase Assay Kit Albumin EIA Kit (Bovine) Human Albumin EIA Kit Rat IgE EIA Kit NEW Rat IgG EIA Kit NEW Mouse IgG EIA Kit NEW Human IgG EIA Kit NEW Viral & Microbial Detection Influenza Virus Typing Set Cell biology Apoptosis and Cytotoxicity ApopLadder Ex (Apoptotic DNA Fragment Extraction Kit) ApoPrimer Set In Situ Apoptosis Detection Set LDH Cytotoxicity Detection Kit Premix WST-1 Cell Proliferation Assay System Antibodies Summary of Antibody Clones OTHERS Yatalase Enzyme TBS (Tris-Buffered Saline) Powder TBE (Tris-Borate-EDTA) Powder Phosphate Buffered Salts (PBS) Tris-Glycine-SDS Powder Tris-Glycine Powder Cartilage Staining Kit Adipoinducer Reagent (for animal cell) Peptide Coating Kit CELL BIOLOGY 3 Table of Contents 119

121 Takara s Cell Biology and Antibody Products 3CELL BIOLOGY Extracellular Matrix Laminin Procollagen Osteocalcin, Gla-OC and Glu-OC Fibronectin Vitronectin Bone Matrix Osteonectin Dentin Matrix Protein Takara's Cell Biology and Antibody Products Human E Cadherin Cell Membrane Cadherin β Catenin σ - 5 Integrin α Catenin β1 Integrin Rat and Mouse Heme Oxygenase Von Willebrand factor EGFR σ - 2 Integrin Calpain FAK β3 Integrin Calpastatin Cell Membrane Endoplasmic Reticulum Takara Products = EIA Kit = Antibodies = Reagent 120

122 Takara s EIA Kit The Takara EIA Kit is a complete system for measuring specific molecules present in biological samples such as plasma, serum, urine and cultured cell extracts. The majority of these kits are based on a sandwich-type enzyme immunoassay (EIA), which utilizes a pair of monoclonal antibodies, each recognizing a different epitope on the antigen molecule. Quantitative measurement of antigen binding is mediated by enzymatic color formation in 96-well microtiter plates. Principle One of the antibodies in the pair is supplied pre-labeled with peroxidase (POD), and the other clone is precoated onto the microtiter plate. Specimens (or standard solutions) are incubated in the coated microtiter plate wells, and the antigen is captured onto the plate. After washing the plate, the labeled antibody is added to the wells and another incubation step is performed, tagging the bound antigen with PODlabeled antibody. Reaction between the plate-bound POD and chromogenic substrate (H 2 O 2, and TMBZ) generates production of colored reaction product. The quantity of reaction product can be measured by a plate reader and is proportional to the amount of antigen present in the specimens or standards. Assay Duration The incubation and detection steps on pre-coated microtiter plates require approximately 3 hours. Total Assay Capacity 96 assays Assay Capacity for Test Sample If all assays (including standards) are run in duplicate, 48 reactions are possible. Test Sample Type These assays can generally be conducted on human serum, plasma, or urine; culture supernatants; or cell extracts. Recommended sample types may vary by kit. Specimen Volume Required If each test sample is run in duplicate, approximately 210 µl (i.e., 100 µl per assay plus 10 µl for each sample transfer) is required. One-step sandwich EIAs require 50 µl. Standard Curves Since conditions may vary from assay to assay, a standard curve must be established for every run. Refer to user manual for more information if culture supernatants are to be measured. Store at 2 to 8 C CELL BIOLOGY 3 1st incubation 2nd incubation Antigen Immobilized antibody Two-Step Sandwich-Type EIA Labeled antibody E Substrate Color formation E Sandwich complex One-Step Sandwich-Type EIA Enzyme Immunoassay Kits 121

123 Summary of Takara EIA Kits 3CELL BIOLOGY Product Cat.# Assay range Sensitivity Principle Sample E-cadherin MK ,700 ng/ml 84.4 ng/ml Precoated Two-step sandwich 96 assays GMP-140 MK ng/ml 20 ng/ml Precoated Two-step sandwich 96 assays PIP (Procollagen type I C-Peptide) MK ng/ml 10 ng/ml Precoated One-step sandwich 96 assays RetroNectin EIA Kit MK ng/ml 3.1 ng/ml Precoated Two-step sandwich 96 assays Fibronectin MK ng/ml 25 ng/ml Precoated Two-step sandwich 96 assays Vitronectin MK ng/ml 5 ng/ml Precoated One-step sandwich 96 assays Laminin MK ng/ml 5 ng/ml Precoated Two-step sandwich 96 assays Gla-Osteocalcin MK ng/ml 0.5 ng/ml Precoated Two-step sandwich 96 assays Undercarboxylated-Osteocalcin MK ng/ml 0.25 ng/ml Precoated Two-step sandwich 96 assays Rat Gla-OC Competitive MK ng/ml 30 ng/ml Precoated Two-step sandwich 96 assays Rat Glu-OC Competitive MK ng/ml 10 ng/ml Precoated Two-step sandwich 96 assays Rat Gla/Glu-Osteocalcin MK147 Gla-OC: ng/ ml; Glu-OC: ng/ml Summary of Takara EIA Kits The EIA Kits are for research use only. They should not be used in diagnostic or therapeutic procedures ng/ml; ng/ml Precoated Two-step sandwich 1 set Human Gla-Osteocalcin MK ng/ml 0.2 ng/ml Precoated Two-step sandwich 96 assays Influenza Virus Typing Set MK421 Equivalent to PAP method Avidin-biotin method 96 assays Rat Heme Oxygenase-1 MK ng/ml ng/ml Precoated Two-step sandwich 96 assays Mouse Heme Oxygenase-1 MK ng/ml ng/ml Precoated Two-step sandwich 96 assays Glycocalicin MK ng/ml 10 ng/ml Precoated Two-step sandwich 96 assays Guidelines to Select an Osteocalcin EIA Kit OC Assay Kit (Cat.#) Gla-OC EIA Kit (MK111) Two antibodies for sandwich EIA Glu-OC EIA Kit (MK118) Two antibodies for sandwich EIA Rat Gla-OC High Sensitive EIA Kit (MK126) Two antibodies for sandwich EIA Rat Glu-OC High Sensitive EIA Kit (MK146) Two antibodies for sandwich EIA Human Gla-OC High Sensitive EIA Kit (MK128) Two antibodies for sandwich EIA Pig Gla-Osteocalcin EIA Kit (MK139) Two antibodies for sandwich EIA Pig Glu-Osteocalcin EIA Kit (MK149) Two antibodies for sandwich EIA Mouse Gla-OC High Sensitive EIA Kit (MK127) Two antibodies for sandwich EIA Mammal samples suitable for each kit Serum sample Urine sample Human, Bovine, Rabbit, Dog, Sheep, Goat No mammal samples can be measured. 1 Human, Bovine, Rabbit, Pig, Sheep, Goat, Monkey Rat Rat Human Pig Pig Mouse Human 2 on other species have not been tested. Rat Rat N/A Pig Pig N/A 1 Urinary osteocalcin is typically to be fragmented. Since the epitope regions of two antibodies for the EIA sandwich method in TAK MK111 kit are widely spaced, this kit is not appropriate to measure fragmented Gla-OC. 2 Since the recognized epitopes of the two antibodies used in the TAK MK118 EIA Kit are neighboring, this kit is also suitable for measurement of fragmented Glu-OC in urinary samples

124 Pig Gla-Osteocalcin and Glu-Osteocalcin EIA Kits Pig Gla-Osteocalcin EIA Kit Cat.# MK assays Pig Glu-Osteocalcin EIA Kit Cat.# MK assays Application Quantitative Determination of Porcine Gla-type or Glu-type osteocalcin in porcine serum, plasma, ascite fluid, urine, and cell culture supernatant Concurrent measurement of Gla-osteocalcin and Glu-osteocalcin may be achieved with the Pig Gla-Osteocalcin EIA Kit (Cat. #MK139) and Pig Glu-Osteocalcin EIA Kit (Cat. #MK149) to monitor both bone formation and bone resorption, based on a relative evaluation of Gla/Glu-osteocalcins. Osteocalcin (OC) comprises 49 amino acids, including 2 to 3 γ-carboxyglutamate residues (Gla), and has a molecular weight of approximately 5,900. It is known as a vitamin K-dependent calcium-binding non-collagen protein. Osteocalcin is an osteoblast-specific marker as it is produced only by osteoblasts. It is also a hormone that plays an important role in glucose metabolism. While carboxylated osteocalcin (coc) is the active form of the protein in bone metabolism, undercarboxylated osteocalcin (ucoc) is the active form of the protein in glucose metabolism. Animal Model for Bone Remodeling In bone formation, the dynamic osteogenesis in growing young animals is called modeling (new construction). In matured animals beyond the growing phase, bone morphology undergoes no apparent changes and remains stable, but a certain percentage of the bone are constantly being replaced. This process is called remodeling (reconstruction). Efficacy assessments of osteoporosis drugs require the use of 2 types of animal models to allow evaluation for bone modeling and remodeling. Rodents, such as mouse and rat, are commonly used animal models to evaluate modeling, while monkey and miniature pig are used frequently to evaluate remodeling. These animal models are essential in the developments and efficacy assessments of potential therapeutic agents. Quantitative Osteocalcin Analysis The Pig Gla-Osteocalcin EIA Kit (Cat. #MK139) is a quantitative kit that enables specific and highly sensitive assay of porcine Gla-osteocalcin that exhibits a potential to osseointegration (active osteocalcin). The capture antibody is a plate-bound solidphased monoclonal antibody that specifically recognizes the Gla residue at position 17 on osteocalcin. It is paired with a labeled monoclonal antibody for detecting porcine osteocalcin. The Pig Glu-Osteocalcin EIA Kit (Cat. #MK149) is a quantitative kit that enables specific and highly sensitive assay of decarboxylated osteocalcin released from porcine bone tissues by enzymes in osteoclasts or undercarboxylated Glu-osteocalcin (ucoc; inactive osteocalcin) produced by osteoblasts. The capture antibody is a plate-bound solid-phased monoclonal antibody that specifically recognizes the Glu residues at positions 21 and 24 on osteocalcin. It is paired with a labeled monoclonal antibody for detecting porcine osteocalcin. Kit Components MK139 and MK149 Antibody Coated Microtiter plate 1 plate (8 wells 12 strips) Antibody-POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (11 ml 2) Substrate Solution 1 vial (12 ml) Performance Characteristics Pig Gla-Osteocalcin EIA Kit (Cat.# MK139) Range of assay: ng/mll Sensitivity: 1.0 ng/ml Assay duration: 2.5 hours Specificity: This kit specifically measures Porcine Gla-OC. Species cross-reactivity: Reactivity with bovine and rat Gla-OC. Test specimen type: porcine serum, plasma, ascite fluid, urine, and cell culture supernatant Specimen volume required: 100 µl/assay well is required. Pig Glu-Osteocalcin EIA Kit (Cat.# MK149) Range of assay: ng/mll Sensitivity: 0.5 ng/ml Assay duration: 2.5 hours Specificity: This kit specifically measures Porcine Gla-OC. Species cross-reactivity: Reactivity with bovine and rat Glu-OC. Test specimen type: porcine serum, plasma, ascite fluid, urine, and cell culture supernatant Specimen volume required: 100 µl/assay well is required. Notes: These products cross-react slightly with bovine antigens, any bovine-serum supplemented medium in a cultured sample may interfere with the assay. Switch to a serum-free medium. These products cross-react slightly with rat antigens, resulting in low sensitivity. They are therefore unsuitable for assaying samples of rat origin. We recommend Rat Gla- Osteocalcin High Sensitive EIA Kit (Cat. #MK126) or Rat Glu-Osteocalcin High Sensitive EIA Kit (Cat. #MK146) for assaying rat samples. 4 C CELL BIOLOGY

125 3CELL BIOLOGY Human Gla-Osteocalcin High Sensitive EIA Kit Human Gla-Osteocalcin High Sensitive EIA Kit Cat.# MK assays Highly Sensitive Quantitative Determination of Gla-osteocalcin in the Supernatant of Human Cell Cultures or in Human Biological Samples Use to Differentiate Human and Bovine Osteocalcins Human Gla-OC High Sensitive EIA Kit enables direct assay of human osteocalcin in the culture supernatant of osteoblasts or differentiated osteoblasts from bone marrow or mesenchymal stem cells cultured in a bovine serum-containing medium. This kit is designed to precisely differentiate bovine and human osteocalcins, using a capture antibody-coated plate with a human osteocalcin-specific monoclonal antibody that recognizes the distinct difference in the amino acids at positions 3 and 4 from the N-terminus. Osteocalcin, also known as bone γ-carboxyglutamate protein, is a vitamin K- and vitamin D- dependent, calcium-binding, non-collagenous protein. Osteocalcin is produced only by osteoblasts and their dental counterpart, odontoblasts, and is an indicator of bone metabolism. The carboxylated form of osteocalcin, in particular, is an indicator of bone formation. The detection antibody in this kit specifically recognizes the γ-carboxyglutamate (Gla) at position 17, which preferentially measures the Gla-type (active type) osteocalcin that binds to the bone matrix (primarily hydroxyapatite). Kit Components MK128 Antibody Coated Microtiter plate 1 plate (8 wells 12 strips) Antibody-POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (11 ml 2) Substrate Solution 1 vial (12 ml) Performance Characteristics Range of assay: ng/ml Sensitivity: 0.2 ng/ml Assay duration: 3.5 hours Specificity: This kit specifically measures human Gla-OC Test specimen type: Human cell culture supernatants and extracts and human serum Specimen volume required: 100 µl/assay well 4 C Enzyme Immunoassay Kits Mouse Gla-Osteocalcin High Sensitive EIA Kit Mouse Gla-Osteocalcin High Sensitive EIA Kit Cat.# MK assays Application Highly Sensitive Quantitative Determination of Gla-type osteocalcin (Mouse Gla-OC) in mouse biological samples and osteoblast cultures The Mouse Gla-Osteocalcin High Sensitive EIA Kit is an quantitative kit that enables specific and highly sensitive assay of mouse Gla-osteocalcin that exhibits a potential to osseointegration (active osteocalcin). The capture antibody (plate-bound antibody) is a plate-bound solid-phased rat monoclonal antibody that specifically recognizes the C-terminal region of mouse osteocalcin. It is paired with labeled antibody a monoclonal antibody for detecting osteocalcin with Gla residues. Because mouse osteocalcin has C terminal region sequences that differ from those in humans, cattle and other large animals, it is possible to measure mouse osteocalcin without any cross-reaction with bovine antigens through capture of the antigen with antibodies recognizing a C-terminal epitope. Therefore, one can monitor the process of osteoblastic cell differentiation from pluripotent cells such as mouse ES and ips cells without interference from bovine serum included in the culture medium. Furthermore, this kit can be used to carry out high-sensitivity measurements on not only cell culture supernatants, but also samples of mouse blood and bodily fluids. Osteocalcin (OC) comprises 49 amino acids, including 2 to 3 γ-carboxyglutamate residues (Gla), and has a molecular weight of approximately 5,900. It is known as a vitamin K-dependent calcium binding non-collagen protein. Osteocalcin is an osteoblast-specific marker as it is produced only by osteoblasts. The Gla-osteocalcin, in particular, is a marker of osteogenesis. In bone formation, the dynamic osteogenesis in growing young animals is called modeling (new construction). In matured animals beyond the growing phase, bone morphology undergoes no apparent changes and remains stable, but a certain percentage of the bone are constantly being replaced. This process is called remodeling (reconstruction). For mice of approximately 8 weeks of age, measurement is possible using a sample dilution of 10 to 20-fold, enabling monitoring of Gla-type osteocalcin concentration even when it is only possible to collect a minimal volume of mouse serum. Kit Components MK127 Antibody Coated Microtiter plate 1 plate (8 wells 12 strips) Antibody-POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (11 ml 2) Substrate Solution 1 vial (12 ml) Performance Characteristics Range of assay: ng/ml Sensitivity: 0.5 ng/ml Assay duration: 2.5 hours Specificity: This kit specifically measures mouse Gla-OC Test specimen type: Mouse cell culture supernatants and extracts, serum, plasma and ascites Specimen volume required: 100 µl/assay well 4 C 124

126 Gla-type Osteocalcin (Gla-OC) EIA Kit (Precoated) Gla-type Osteocalcin (Gla-OC) EIA Kit (precoated) Cat.# MK assays Application Quantitative Measurement of Human, Bovine, Rabbit, Sheep, Dog or Goat Carboxylated Osteocalcin Osteocalcin (OC), also known as g-carboxyglutamic acid protein, is a small (5.9 kda), vitamin K-dependent, hydroxyapatite (Ca 2+ )-binding protein synthesized exclusively by osteoblasts and odontoblasts. This tissue-specific expression of OC makes it an excellent indicator of overall cell activity for bone formation. Three g-carboxyglutamic acid (Gla) residues at positions 17, 21, and 24 of the protein are responsible for binding calcium. Calcium-binding is, in turn, required for such biological activities as activation of the blood coagulation cascade. Thus, Gla-OC likely represents the active form of the protein, while Glu-OC, which has weak binding affinity to hydroxyapatite, represents the inactive form. Serum OC levels correlate well with bone formation rates and, thus, enzyme immunoassay (EIA) measurements of carboxylated OC (Gla-OC) vs. decarboxylated OC (Glu-OC) can be used for preclinical and clinical evaluation studies. EIA systems may be more informative than conventional assays, which cannot differentiate between active and inactive forms. The Gla-Type Osteocalcin EIA Kit is a 96-well in vitro enzyme immuno assay kit for quantitative determination of human Gla-OC in serum, cultured cell extracts, cell culture supernatants, and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal Gla-OC antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of carboxylated osteocalcin using a two-step method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled Gla-OC antibody. A substrate is added, and the reaction between POD and the substrate (TMBZ in presence of H 2 O 2 ) results in Application Quantitative Measurement of Human, Bovine, Rabbit, Sheep, Pig and Goat Undercarboxylated Osteocalcin The Undercarboxylated (Glu-OC) Osteocalcin EIA Kit is a 96-well in vitro enzyme immunoassay kit for quantitative determination of human Glu-OC in serum, plasma, urine, cultured cell extracts, cell culture supernatants, and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal Glu-OC antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of carboxylated osteocalcin using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled Glu-OC antibody. A substrate is added, and the reaction between POD and the substrate (TMBZ in presence of H 2 O 2 ) results in development of a colored product. The amount of sample Glu-OC is determined by measuring absorbance using an EIA plate reader. Accurate Glu-OC sample concentrations can be determined by comparing sample absorbance values with the absorbance obtained for the standard plotted on a standard curve. a color development. The amount of sample Gla-OC is determined by measuring absorbance using an EIA plate reader. Accurate Gla-OC sample concentrations can be determined by comparing their specific absorbances with the absorbance obtained for the Standard plotted on a standard curve. Kit Components MK111 Antibody coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard 1 vial (8 ng) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 0.5 ng/ml Assay duration: 3.5 hours Specificity: Human Gla-OC. No detectable cross reactivity with undercarboxylated (Glu) OC. Species cross-reactivity: Reactivity with bovine, rabbit, sheep, dog, and goat Gla-OC. No reactivity: with mouse and rat Gla-OC. : for other species have not been tested. Test specimen type: Serum, plasma, culture supernatants, or cell extracts. Sample volume per well: 100 µl 2 8 C Undercarboxylated (Glu-OC) EIA Kit Undercarboxylated Osteocalcin (Glu-OC) EIA Kit (precoated) Cat.# MK assays Kit Components MK118 Antibody coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard 1 vial (8 ng) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 0.5 ng/ml Assay duration: 3.5 hours Specificity: Human Glu-OC. Species cross-reactivity: Reactivity with bovine, rabbit, sheep, pig and goat Glu-OC. No reactivity: with mouse and rat Glu-OC. : for other species have not been tested. Test specimen type: Serum, plasma, urine, culture supernatants, or cell extracts. Sample volume per well: 100 µl 2 8 C CELL BIOLOGY 3 Enzyme Immunoassay Kits 125

127 Gla-Type Osteocalcin and Undercarboxylated (Glu-OC) EIA Kit Gla-type Osteocalcin EIA Kit Undercarboxylated Osteocalcin EIA Kit CELL BIOLOGY Amount osteocalcin in serum and citrate plasma of the same subject Cross reactivity Amount of osteocalcin in urine Gla-OC (ng/ml) 10 0 Serum Average ng/ml SD n=35 (age average 29) With synthetic human undercarboxylated osteocalcin-0% Not detected Citrate plasma ng/ml Figure 3 Amount of Gla-type osteocalcin in normal serum and normal citrate plasma OC (ng/ml) Undercarboxylated Serum Citrate plasma Average ng/ml ng/ml SD n=35 (age average 29) Figure 4 Amount of undercarboxylated osteocalcin in normal serum and normal citrate plasma With bovine bone osteocalcin-1.7% With human bone osteocalcin-5.0% With synthetic human Gla-type osteocalcin-3.8% Detected in urine of lactate woman (Figure 5-a) and of little children (Figure 5-b) Considerably correlative with deoxypyridinolin (Comparison Figure 5-b with Figure 5-c) Enzyme Immunoassay Kits (ng/mg.cr) Glu-OC Urinary Average n= SD Age control p<0.001 p<0.001 Pregnant p=0.534 Lactate Figure 5-a Urinary Undercarboxylated OC value in normal, pregnant and lactate women Glu-OC (ng/mg.cr) Age (years) Figure 5-b Urinary Undercarboxylated OC/Cr in normal person (nmol/mg.cr) Deoxypridinolin Age (years) Figure 5-c Urinary deoxypridinolin/cr in normal person 126

128 Rat Gla/Glu-Osteocalcin High Sensitive EIA Kit Rat Gla-Osteocalcin High Sensitive EIA Kit Cat.# MK assays Rat Glu-Osteocalcin High Sensitive EIA Kit Cat.# MK assays Gla/Glu-Osteocalcin High Sensitive EIA Kit (Rat) Cat.# MK147 1 set Application Quantitative Determination of Gla-Osteocalcin or Glu-Osteocalcin in Rat-Derived Biological Samples Rat Gla-Osteocalcin High Sensitive EIA Kit (Cat.# MK126) is a 96-well format sandwich-type EIA kit, which uses a rat osteocalcin C-terminus-specific antibody as a capture antibody on a solid-phase plate. This antibody has minimal cross reactivity with bovine, human and rabbit osteocalcin. An enzyme-labeled antibody (GlaOC4-30) specific to Gla-OC is used as the detection antibody, allowing this kit to detect Gla-osteocalcin with very high sensitivity. As such, this EIA kit is sensitive enough to detect even trace levels of rat osteocalcin produced in supernatants of cells cultured in fetal calf serum-supplemented medium. Rat Glu-Osteocalcin High Sensitive EIA Kit (Cat.# MK126) is a 96-well format sandwich-type EIA kit, in which rat osteocalcin C terminal region recognition specific antibody is used as the capture antibody on a solid plate and a monoclonal antibody that specifically recognizes the Glu residues that straddle positions 21 and 24 of osteocalcin used as the detection antibody. This kit makes high-sensitivity measurement of minor antigens and maintenance of stable reproducibility possible. Moreover, as it has the same capture antibody as the Rat Gla-Osteocalcin High Sensitive EIA Kit (Cat. # MK126), the kits may be used together for simultaneous Gla/Glu detection (Cat.# MK147), thereby making simultaneous monitoring of bone formation and resorption possible. Specifically produced by only osteoblasts, osteocalcin has been used as an osteoblast marker. Rat osteocalcin consists of a total of 50 amino acids. Human, bovine, rabbit, and other species have a osteocalcin with 49 amino acids. The three glutamate residues at positions 17, 21, and 24 of the amino acid chain are carboxylated, forming a calcium pocket that allows osteocalcin to bind to bone matrix. Osteoblasts generally produce osteocalcin with all three glutamate residues carboxylated (Gla-OC), allowing the protein to bind with bone matrix. During bone metabolism, osteocalcin is released from bone matrix through the actions of various enzymes, including one produced by osteoclasts. Most of the three glutamate residues are decarboxylated on osteocalcin (Glu-OC) when it is released into blood MK126 Rat Gla-OC EIA Kit from bone. Therefore, osteocalcin is present in blood in both Gla and Glu forms and is made up of a wide range of molecular species, from full-length to fragmented molecules. Kit Components MK126 Antibody Coated Microtiterplate 1 plate (8 wells 12 strips) Antibody - POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (2 11 ml) Substrate Solution 1 vial (12 ml) Performance Characteristics Rat Gla-Osteocalcin High Sensitive EIA Kit (Cat.# MK126) Range of assay: ng/mll Sensitivity: 0.25 ng/ml Assay duration: 2.5 hours Specificity: This kit specifically measures Rat Gla-OC. Test specimen type: rat serum, ascites, cell culture supernatant, and cell extracts Specimen volume required: 100 µl/assay well is required. Rat Glu-Osteocalcin High Sensitive EIA Kit (Cat.# MK146) Range of assay: ng/ml Sensitivity: ng/ml Assay duration: 2.5 hours Specificity: This kit specifically measures Rat Glu-OC. Test specimen type: rat serum, ascites, cell culture supernatant, and cell extracts Specimen volume required: 100 µl/assay well is required. 4 C Simultaneous Measurement of Glu-OC and Gla-OC in Rat Serum Both types of osteocalcin (Glu- type and Gla- type) were monitored in the blood serum of young or aged rats over several weeks. The values in the table are reduced concentrations multiplied by the dilution ratio. The Gla/Glu-Osteocalcin High Sensitive EIA Kit (Rat; MK147) may be used for this analysis. MK146 Rat Glu-OC EIA Kit Age (Weeks) Dilution Male No. 1 Male No. 2 Female No. 1 Female No. 2 Dilution Male No. 1 Male No. 2 Female No. 1 Female No fold fold fold CELL BIOLOGY 3 Enzyme Immunoassay Kits 6 60-fold fold fold fold Age (Weeks) Dilution Female No. R1 Female No. R2 Dilution Female No. R1 Female No. R fold fold fold fold

129 3CELL BIOLOGY Osteoblast-Inducer Reagent (for animal cell) Osteoblast-Inducer Reagent (for animal cell) Cat.# MK430 1 kit Bone tissue consists of osteoclasts, which are responsible for bone resorption, and Kit Components Ascorbic Acid 5 ml osteoblasts and osteocytes, which are responsible for bone formation. These cell Hydrocortisone 1 ml groups work in concert to maintain bone strength and elasticity. β-glycerophosphate 2 5 ml This product contains a set of osteoblast-inducer reagents, including hydrocortisone, β-glycerophosphate, and ascorbic acid. These reagents induce the efficient differentiation of bone marrow-derived cells and adipose-derived stem cells (mesenchymal stem cells) into osteoblasts when added to culture medium. 20 C Enzyme Immunoassay Kits 128

130 TRACP & ALP Double Stain Kit TRACP & ALP Double Stain Kit Cat.# MK reactions Application Differential Visualization of TRACP and ALP Activities in Bone-Related Cells The TRACP & ALP Double Stain Kit allows differential staining of osteoblast and osteoclast cells (bone-related cells). With this kit, chromogenic substrates for alkaline phosphatase, an enzyme marker of osteoblasts, and tartrate-resistant acid phosphatase, an enzyme marker of osteoclasts, are combined with a reagent for nuclear staining that provides visualization of multinucleated osteoclasts. Both acid and alkaline phosphatase activities in the cells can be stained simultaneously for comparison. Bone metabolism is mutually balanced between bone formation by osteoblasts and bone absorbsion by osteoclasts. Simultaneous detection of both enzyme markers provides a means to study the differentiation of bonerelated cells. The kit substrates are provided as premixed reagents for ease of preparation. Sufficient reagent is supplied for staining approximately 5 culture plates (24 well size). Kit Components MK300 Fixation solution Citrate buffer (ph 5.4) containing 60% acetone and 10% methanol 30 ml Sodium tartrate 0.5 M sodium tartrate buffer (ph 5.2) 4 ml Substrate for ACP (premixed) NABP/FRVLB 3 vials* Substrate for ALP (premixed) BCIP/NBT 3 vials* Nuclear stain Methyl green (in acetic acid solvent) 10 ml *Resuspend the lyophilized substrate in each vial with 10 ml distilled water. 20 C CELL BIOLOGY 3 Figure 1. Human bone marrow mononuclear cells were cultured in the presence of different additive substances, Macrophage Colony Stimulating Factor (+M-CSF) and +Vitamin D3. TRACP activity staining was carried out on day 9 of cultivating, after differentiation had occurred. Figure 2. Human bone marrow mononuclear cells were cultured in the presence of Macrophage Colony Stimulating Factor (+M=CSF). ALP activity staining was carried out on day 9 of cultivating, after differentiation had occurred. Enzyme Immunoassay Kits TRACP & ALP Assay Kit TRACP & ALP Assay Kit Cat.# MK reactions Application Detection of Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) TRACP & ALP Assay Kit has been designed for simple and quick detection of ACP (Acid phosphatase) and ALP (Alkaline phosphatase) by utilizing pnpp (p-nitro-phenyl phosphate) substrate. This kit allows the simultaneous detection of 2 enzymes which are involved in bone metabolism, TRACP and ALP which is an enzyme marker of osteoblasts. The addition of tartaric acid into the ACP. ALP allows for the detection of TRACP is a marker of osteoclasts activity and enzyme marker of osteoclast. Since this kit utilizes an aqueous substrate, it allows for quick quantification of the enzyme activity by measuring the absorbance of the reactant. In addition to this kit, a TRACP & ALP Double-Staining Kit (Cat.# MK300) is also available for the assay using a non-soluble substrate. The appropriate kit can be selected depending on an assay of interest. Kit Components MK301 pnpp (p-nitro-phenyl phosphate) substrate 5 vials 24 mg Extraction solution 2 vials 11 ml Sodium tartrate solution 4 ml Buffer for ACP 30 ml Buffer for ALP 30 ml Microplate (96-well)* 1 plate *The plate is reusable after soaking in 1% sodium hypochlorite solution overnight. 4 C 129

131 3CELL BIOLOGY Enzyme Immunoassay Kits Procollagen Type I C-Peptide (PIP) EIA Kit (Precoated) Procollagen Type I C-Peptide (PIP) EIA Kit (Precoated) Cat.# MK assays (One-step sandwich-type EIA) Application Quantitative Measurement of Human, Bovine, Dog or Horse PIP Collagen types I, II, III, IV and V are synthesized as precursor molecules called procollagens. These precursor molecules contain additional peptide sequences, termed propeptides, at both the amino-terminal and the carboxy-terminal ends. Propeptides function to facilitate the winding of procollagen molecules into a triplehelical conformation within the endoplasmic reticulum. The propeptides are cleaved from the collagen triple helix molecule during its secretion, after which the triple helix collagens polymerize into extracellular collagen fibrils. Thus, the amount of the free propeptides stoichiometrically reflects the amount of collagen molecules synthesized. In particular, procollagen type I carboxy-terminal peptide (PIP) has been used as a reference for studying the correlation of collagen levels with certain health disorders, including bone diseases, alcoholic liver diseases, liver cirrhosis, and scirrhous (Borrmann type IV) adenocarcinoma of the stomach. The Procollagen Type I C-Peptide EIA Kit is a 96-well format in vitro enzyme immunoassay for the quantitative determination of human, bovine, or canine PIP in plasma, serum, cultured cell extracts, cell culture supernatants, and other biological samples. This solid phase EIA is based upon a sandwich design that utilizes two mouse monoclonal PIP antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of PIP using a one-step incubation method. Samples and POD-anti-PIP are simultaneously added to the wells of the plate and incubated. During incubation, PIP binds to the antibody coating the plate, and is then bound by POD-anti-PIP on its opposite side. A substrate is added, and the reaction between POD and the substrate (H 2 O 2, TMBZ) results in a color development. The amount of sample PIP is determined by measuring absorbance using an EIA plate reader. Accurate PIP sample concentrations can be determined by comparing their specific absorbance in the sample with the absorbance obtained for the Standards plotted on a standard curve. 2 Kit Components MK101 Antibody coated microtiter plate 1 plate (8 well 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 10 ng/ml Assay duration: 3.5 hours Specificity: Human PIP. Does not recognize fibronectin, vitronectin, laminin, collagen type I, or collagen type III. Species cross-reactivity: Recognizes bovine, dog and horse PIP. Does not recognize mouse PIP. Other species have not been tested. Test specimen type: Serum, plasma, culture supernatants (serum-free), and cell extracts Sample volume per well: 20 µl 2 8 C 492 at Absorbance nm ,000 Procollagen type I C-Peptide (ng/ml) Typical Procollagen Standard Curve. A stock solution containing 640 ng PIP/mL was used to generate a standard curve. A dilution series was prepared by mixing the standard solution and sample diluent. Standard curves should be generated for each new assay

132 Application: Using the Procollagen Type I C-Peptide EIA Kits Medium (FCS) 253J (kidney cancer) 769P (kidney cancer) 19PC (prostatic cancer) OST (osteosarcoma) MG-63 (osteosarcoma) HMV-II (melanoma) Lu-65 (lung cancer) MKN-45 (gastric cancer) KB (ears and throat cancer) HT-1080 (fibrosarcoma) (ng/ml) Conc. of PIP (ng/ml) 1, MG63 cells TGF-β (+) TGF-β ( ) CELL BIOLOGY IMR-90 (fetal lung) HFL-1 (fetal lung) FLOW (fetal lung) Measurement of PIP in cultured supernatant of several cell lines (background levels in media will differ between FCS batches) Culture days Stimulation of Collagen Synthesis by TGF-β 3 Application Example. Measurement of PIP in cultured supernatant of several cell lines. Note: measurement with this kit may be shifted if it is performed with samples which include animal serum such as Fetal Bovine Serum and Horse Serum. It is recommended to perform the measurement under serum-free conditions. Example Experiment: Stimulation of Collagen Synthesis by TGF-beta. This kit uses clone PC8-7 (M012) as the solid-phase antibody and clone PC5-5 (M011) as labeled antibody. These monoclonal antibody clones were raised against purified human procollagen type I carboxy-terminal propeptides. RetroNectin EIA Kit RetroNectin EIA Kit Cat.# MK140 1 kit Application Quantitative Determination of Release from RetroNectin-Coated Plates and Residual RetroNectin Levels This kit is a sandwich ELISA using as the capture antibody a plate-coated RetroNectin-specific monoclonal antibody that does not cross-react with human fibronectin at all. Its binding is not inhibited by the presence of human blood components, allowing easy monitoring of the level of RetroNectin. This kit allows highly sensitive quantitative determination of release from RetroNectin-coated dishes and permits detection of RetroNectin present in cryopreservation solution of cells cultured in medium containing RetroNectin Reagent. RetroNectin Reagent (recombinant human fibronectin CH-296), a recombinant protein, is composed of 3 functional domains: human fibronectin cell-binding (C-domain), heparin-binding domain (H-domain) and CS-1 sequence. A combination of a recombinant retroviral vector and RetroNectin Reagent is frequently used for gene transfer into mammalian cells. This protocol, in which the RetroNectin molecule brings the retroviral vector that binds to the H-domain into close proximity with the animal cell that has an affinity for the C-domain, can dramatically increase transduction efficiency. In addition, RetroNectin has been used to aid lymphocyte expansion after it was shown to be an effective matrix for coating apparatuses used in ex vivo expansion of lymphocytes. Performance Range of assay: ng/ml Sensitivity: 3.1 ng/ml Assay duration: 2.5 hrs. for assay protocol Specificity: This kit specifically measures RetroNectin Reagent. Test specimen type: Cryopreservation Solution or washing solution of cells cultured on RetroNectin Reagent coated plate as samples. Specimen volume required: 100 µl/ assay well. Kit Components MK140 Antibody Coated Microtiter Plate Anti-RetroNectin monoclonal antibody plate : 1 plate (96 wells: 8 well 12 strips) Antibody-POD Conjugate: Peroxidase-labeled anti-retronectin antibody (lyophilized) 1 vial (for 1 11 ml) Standard RetroNectin standard: 200 ng (lyophilized) 1 vial (for 1 11 ml) Sample Diluent: 2 vials (2 11 ml) Substrate Solution (TMBZ): 3,3,5,5 -tetramethyl benzidine solution 4 C Enzyme Immunoassay Kits 131

133 3CELL BIOLOGY Fibronectin EIA Kit (Precoated) Fibronectin EIA Kit (Precoated) Cat.# MK assays (Two-step sandwich-type EIA) Application Quantitative Measurement of Human Fibronectin in Serum The adhesive glycoprotein fibronectin (FN) is a 440 kda protein found on cell surfaces, in the extracellular matrix, and in plasma. FN is involved in many cellular processes including cell-to-substrate adhesion, cell migration, and regulation of cell morphology. Increased amounts of fragmented FN or FN-like protein can be observed in serum, plasma, or other biological fluids from cancer patients as compared to levels in normal subjects. The human Fibronectin EIA Kit is a 96-well in vitro enzyme immuno assay kit for quantitative determination of soluble human FN in serum, urine, cell culture supernatants, and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal FN antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of fibronectin using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the PODlabeled FN antibody. A substrate is added, and the reaction between POD and the substrate (H 2 O 2, TMBZ) results in a color development. The amount of sample FN is determined by measuring absorbance using an EIA plate reader. Accurate FN sample concentrations can be determined by comparing their absorbance values with the absorbance obtained for the Standard plotted on a standard curve. Kit Components MK115 Antibody coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 11 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 25 ng/ml Assay duration: 2.5 hours Specificity: Human FN. Does not recognize vitronectin, laminin, collagen type I, or collagen type III. Species cross-reactivity: Does not recognize mouse, bovine, rabbit and porcine FN. Test specimen type: Plasma, serum, urine, culture supernatants, or cell extracts Sample volume per well: 100 µl 2 8 C Enzyme Immunoassay Kits FN(ng/ml) HepG2(SF) HUVEC(SF) HT1080(SF) Colo205(SF) HL60(SF) T24(SF) NSK(SF) MG63(SF) HGH(SF) SQ5(SF) A431(SF) Lu65(SF) OST(SF) BUD-8(+S) KM101(SF) KM101(+S) SIRC(SF) SIRC(+S) D-17(SF) D-17(+S) Flamnion(SF) Flamnion(+F) N.F.(SF) N.F.(+S) CPAE(SF) CPAE(+S) Cell Strain Application Example: Fibronectin Levels in Cell Culture Supernatants The amount of Fibronectin in the supernatant of various cells cultured in 10% FCS/RPMI 1640 medium or serum-free medium (Ultradoma PF) was measured. The supernatant assayed without dilution. Fetal calf serum does not inhibit this assay system. +S: 10% FCS/RPMI 1640 SF: serum free medium (2 days after the change from the medium containing serum) Abbreviations: HepG2: human hepatocellular carcinoma SQ5: human lung squamous cell carcinoma HUVEC: human umbilical vein endothelial cell A431: human spidermoid carcinoma HT1080: human fibrosarcoma Lu65: human lung carcinoma Colo205: human adenocarcinoma OST: human osteosarcoma HL60: human promyelocytic leukemia KM101: human bone marrow stromal cell T24: human bladder transitional-cell carcinoma SIRC: rabbit cornea cell NSK: human normal skin cell D-17: canine osteogenic sarcoma MG63: human osteosarcoma Flamnion: human amnion cell HGH: human Girardi heart N.F.: human normal fibroblast cell CPAE: bovine vein endothelial cell Fibronectin-Related Peptides Fibronectin- Related Peptide GRGDSP Cat.# SP001 1 mg Fibronectin- Related Peptide GRGESP Cat.# SP002 1 mg Purity >98% by HPLC 20 C Form Lyophilized white powder 132

134 Laminin EIA Kit (Precoated) Laminin EIA Kit (Precoated) Cat.# MK assays Application Quantitative Measurement of Human Laminin Laminins (LN) are large multidomain glycoproteins of the extracellular matrix that are important for induction of cell adhesion, growth promotion, mediation of cell communication and enhancement of tumor cell metastasis. LN consists of a, b, and g polypeptide chains that are linked by disulfide bridges to form a characteristic asymmetric cross-structure observed using electron microscopy. LN binds to various components of the basal membrane and is thought to link these components to one another. Cell surface receptors that may play a role in LN-mediated cell adhesion have been isolated from metastatic tumor cells and platelets. Furthermore, serum levels of LN fragments are elevated in patients with hepatic fibrosis, alcoholic liver, hypertension, and several kinds of tumors. The Laminin EIA Kit is a 96-well format in vitro enzyme immunoassay kit for quantitative determination of human laminin in serum, plasma, urine, cultured cell extracts, cell culture supernatants and other biological samples. It is a solid phase sandwich EIA that utilizes two g1-specific mouse monoclonal LN antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of LN using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled LN antibody. A substrate is added, and the reaction between POD and the substrate (H 2 O 2, TMBZ) results in a color development. The amount of sample LN is determined by measuring absorbance using an EIA plate reader. Accurate LN sample concentrations can be determined by comparing absorbance values with the absorbance obtained for the Standard plotted on a standard curve. Laminin Peptides Kit Components MK107 Antibody-coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 5 ng/ml Assay duration: 2.5 hours Specificity: Human LN. Does not recognize human fibronectin, vitronectin, fibrinogen, collagen type I, or collagen type III. Species cross reactivity: Rabbit LN. Does not recognize mouse LN. Other species have not been tested. Test specimen type: Serum, plasma, urine, cultured cell extracts, cell culture supernatants, and other biological fluids Sample volume per well: 50 µl 2 8 C for the complete kit Laminin Peptide YIGSR Cat.# SP003 1 mg Laminin Peptide LGTIPG Cat.# SP004 1 mg Purity >95% by HPLC 20 C Form Lyophilized CELL BIOLOGY 3 Enzyme Immunoassay Kits 133

135 3CELL BIOLOGY Enzyme Immunoassay Kits Vitronectin EIA Kit (Precoated) Vitronectin EIA Kit (Precoated) Cat.# MK assays Application Quantitative Determination of Human Vitronectin Vitronectin (VN) is a major cell adhesion protein found in plasma (at high concentrations of mg/l), platelets, and the extracellular matrix of tissues and atherosclerotic plaques. VN interacts with many different macromolecules, including cell surface integrin receptors, inactive anti-thrombin/thrombin complex, collagen and other extracellular matrix components, plasminogen activator inhibitor type 1, and heparin. Through these interactions, VN contributes in various ways to the regulation of the immune and hemostatic systems. Clinically, it has been noted that a significant decrease of plasma vitronectin levels is observed in patients with liver disorders with high fibroblastic activity, such as liver cirrhosis. The Vitronectin EIA Kit is a 96-well format in vitro enzyme immunoassay kit for quantitative determination of human vitronectin in serum, plasma, cultured cell extracts, cell culture supernatants and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal VN antibodies (one of which is coated on the plate wells, and the other is POD-labeled) for detection of VN using a one-step incubation method. Samples and a POD-labeled VN antibody are simultaneously incubated in the antibody-precoated microtiter plate wells. During the incubation, anti-vn on the well surfaces binds sample VN, and the sample VN is in turn tagged by POD-anti-VN. The reaction between POD and the substrate (H 2 O 2, TMBZ) results in development of a color product. The amount of sample VN is determined by measuring absorbance using an EIA plate reader. Accurate VN sample concentrations can be determined by comparing sample absorbance values with the absorbance obtained for the Standard plotted on a standard curve. Kit Components MK102 Antibody coated microtiter plate 1 plate (8 well 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 5 ng/ml Assay duration: 2.5 hours Specificity: Human VN. Does not recognize human fibronectin, laminin, collagen type I or collagen type III. Species cross-reactivity: Does not recognize mouse VN. Other species have not been tested. Test specimen type: Plasma, serum, cultured cell extracts, cell culture supernatants, or other biological fluids Sample volume per well: 50 µl 2 8 C 134

136 Heparan Degrading Enzyme EIA Kit Heparan Degrading Enzyme EIA Kit Cat. # MK assays Quantitative Measurement of Heparan Degrading Enzyme in Mammals Screening of Heparan Sulfate Degrading Enzyme Inhibitors Heparan sulfate is a ubiquitous complex polysaccharide which is present as a component of mammalian cell membranes. It has been identified in mammalian liver, spleen, skin, placenta and platelets, as well as in cancerous tissue such as melanomas, lymphomas, sarcomas, fibrosarcomas and colon cancer. Furthermore, heparan sulfate has been shown to play an important defensive role against invasion of tumor cells. The activity of heparan sulfate degrading enzyme (also known as heparanese), an enzyme that cleaves heparan sulfate, has been reported to be considerably higher in invasive cancer cells than in normal cells. Thus, the correlation of heparan sulfate degrading enzyme activity with malignancy of cancer cells has gained much interest. Takara s Heparan Degrading Enzyme Assay Kit provides a 96-well non-radioactive assay for the measurement of heparan degrading enzyme activity in cultured cells, tissues, or serum as well as screening of heparan sulfate degrading enzyme inhibitors. This kit is based upon the property of heparin-like molecules to bind bfgf (basic fibroblast growth factor). When heparan sulfate is degraded by heparan sulfate degrading enzyme, it loses its ability to bind to bfgf. Thus, the enzymic activity of a sample can be quantitated by comparing the amount of bfgf-bound undegraded heparan sulfate of the sample to total-bound heparan sulfate of a heparanase-free control. A unique feature of this kit is the use of CBD-FGF, a fusion protein composed of human fibronectin cell-binding domain and human fibroblast growth factor. CBD-FGF is immobilized on the surface of Takara s microtiter plate by an anti-fibronectin antibody containing an epitope in the CBD region. Termed DOC (Domain Oriented Capture), this solid phase attachment method allows a natural 3-dimensional bfgf structure for enhanced accessibility and binding to heparan sulfate. Detection is by a colorimetric method using biotinylated heparan sulfate as the substrate. Glycocalicin EIA Kit, Ver Kit Components MK412 CBD-FGF immobilized microtiter plate 1 plate (8 wells 12 strips) Biotinylated heparan sulfate (in reaction buffer) 1 vial (for 5.5 ml) Reaction buffer for dilution 1 vial (11 ml) Extraction buffer 1 vial (11 ml) Standard (lyophilized) 1 vial (for 250 µl) Avidin POD conjugate (lyophilized) 1 vial (for 11 ml) POD substrate (lyophilized) 1 vial (for 12 ml) Performance Characteristics Range of assay: U/mL Assay sensitivity: 0.1 U/mL Assay duration: 2 3 hours Specificity: All animals Test specimen type: Tissue, cultured cells, or serum Sample volume per well: 25 µl 2 8 C Glycocalicin EIA Kit, Ver Cat. # MK assays Application Quantitative Determination of Human Glycocalicin in Platelet Extract, Plasma and Serum Glycocalicin EIA Kit, Ver is a sandwich-based assay for the quantification of human glycocalicin. It is useful for monitoring platelet membrane destruction in human platelet extracts, serum and plasma and to check membrane integrity of stored platelet concentrates over time. Glycocalicin (GC), a soluble 110 to 135 kda proteolytic fragment of the platelet membrane glycoprotein Ib (GPIb) alpha-chain, is generated by cleavage of GPIb by Calpain (a Ca 2+ -dependent protease) present in platelets. Following cleaveage, glycocalicin is released into the plasma. GPIb binds von Willebrand factor and is also a receptor for thrombin. Glycocalicin is considered to be a specific in vivo marker of platelet membrane damage and can be an indicator of platelet activation. Kit Components MK105 Antibody Coated Microtiter plate Antibody - POD Conjugate (PL52-4) Standard Sample Diluent Substrate Solution (TMBZ) 2 8 C 1 plate (8 well 12 strips) 1 vial (for 11 ml) 1 vial (640 ng) 2 vials (2 11 ml) 1 vial (12 ml) CELL BIOLOGY 3 Enzyme Immunoassay Kits 135

137 3CELL BIOLOGY Human E-cadherin EIA Kit Application Quantitative Measurement of Soluble Human E-cadherin E-cadherin, also known as uvomorulin or Cell-CAM120/80, is one of the subclasses of cadherins (Ca 2+ -dependent cell adhesion molecules found in epithelial cells in a variety of embryonic and adult tissues). Previous studies suggested that unstable or reduced expression of E-cadherin appears to be a common event in cancer progression, such as in lung carcinoma, gastric tumors, hepatocellular carcinoma, breast carcinoma and prostate tumors. A soluble 80 to 84 kda form of E-cadherin, presumed to be a degradation product of the intact 120 kda form, was reported to be elevated in the biological fluids of some cancer patients as opposed to levels found in healthy individuals. The Human E-cadherin EIA Kit is a 96-well format in vitro enzyme immuno assay kit for quantitative determination of soluble human E-cadherin in serum, urine, cell culture supernatants and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal E-cadherin antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of E-cadherin using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled E-cadherin antibody. A substrate is added, and the reaction between POD and the substrate (H 2 O 2, TMBZ) results in development of a colored product. The amount of sample soluble E-cadherin is determined by measuring absorbance using an EIA plate reader. Accurate soluble E-cadherin sample concentrations can be determined by comparing sample absorbance values with the absorbance obtained for the Standard plotted on a standard curve. s t n e i t a Normal subjects Diabetes subjects Hepatitis Leukemia Stomach Kit Components MK117 Antibody-coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ,700 ng/ml Assay sensitivity: 100 ng/ml Assay duration: 3.5 hours Specificity: Human E-cadherin. Species Cross Reactivity: Does not recognize mouse E-cadherin. Other species have not been tested. Test specimen type: Serum, urine, culture supernatants or cell extracts Sample volume per well: 100 µl 2 8 C S N. S N S N P < 0. Enzyme Immunoassay Kits Human E-cadherin EIA Kit Cat.# MK assays P < P < 0 P < C a n c e r P Liver Others E-Cadherin (µg/ml) Levels of soluble E-cadherin in sera of 56 normal subjects, 26 diabetes patients, 4 hepatitis patients and 54 cancer patients. Serum E-cadherin levels were determined by IEMA. The mean level in each group is indicated by a horizontal bar. NS, not significant. E-Cadherin Levels in Various Patients as Measured by the E-Cadherin EIA Kit 136

138 GMP-140 (P-selectin) EIA Kit GMP-140 (P-selectin) EIA Kit Cat.# MK assays Application Quantitative Measurement of Human GMP-140 Granule Membrane Protein 140 (GMP-140, P-selectin, CD62 or PADGEM) is a 140 kda integral membrane adhesion glycoprotein categorized functionally as a member of the LEC-CAM (lectin-epidermal growth factor-complement binding cell adhesion molecules) family or the selectin family. In non-activated conditions, GMP-140 is present in the a-granules of platelets or in endothelial cell Weibel Palade bodies. However, when activated by stimulators such as thrombin or by inflammatory or hemostatic mediators during intravascular inflammation, GMP-140 is rapidly secreted to the plasma membrane to promote adhesion of cells to vascular endothelia or to neutrophils and monocytes at the site of tissue injury. GMP-140 is a useful marker of platelet and endothelium activation in experimental inflammation models. Free GMP-140 molecules present in the circulation may serve to maintain the non-adhesiveness of neutrophils and prevent the development of inflammatory responses. The GMP-140 (P-selectin) EIA Kit is a 96-well format in vitro enzyme immunoassay kit for quantitative determination of human GMP-140 in plasma, serum, cultured cell extracts, cell culture supernatants and other biological samples. It is a solid phase sandwich EIA that utilizes two mouse monoclonal GMP-140 antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of human GMP-140 using a two-step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled GMP-140 antibody. A substrate is added, and the reaction between POD and the substrate (H 2 O 2, TMBZ) results in a color development. The amount of sample GMP-140 is determined by measuring absorbance using an EIA plate reader. Accurate GMP-140 sample concentrations can be determined by comparing their specific absorbances with the absorbance obtained for the Standard plotted on a standard curve. GPDH Activity Assay Kit GPDH Activity Assay Kit Cat.# MK assays Measurement of GPDH Activity Regulating Differentiation of Primary or Established Cell-Line Progenitor Cells into Adipocytes Screening of Differentiation Repressors The enzyme glycerol 3-phosphate dehydrogenase catalyzes a reversible reaction between dihydroxyacetone phosphate (DHAP) and glycerol 3-phosphate, with NAD as a coenzyme. GPDH activity increases significantly during differentiation of progenitor cells into adipocytes and, thus, the activity of this enzyme has been used as an index for monitoring fat synthesis. The Glycerol 3-Phosphate Dehydrogenase Assay Kit is a 96-well microtiter plate assay designed to measure the activity of GPDH. This kit can be used to either screen differentiation repressors or to study mechanisms regulating differentiation of primary or established cell-line progenitor cells into adipocytes. The procedure involves preparation of cell extract by addition of the Enzyme Extraction Buffer, followed by serial dilution of the extract. The serially diluted extract is added to a 96-well plate containing substrate solution, and GPDH activity is calculated by measuring the change (decrease) in absorbance at OD 340 using a microtiter UV plate reader. Kit Components MK112 Antibody-coated microtiter plate 1 plate (8 well 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: 20 ng/ml Assay duration: 3.5 hours Specificity: Human GMP-140. Does not recognize platelet Gp Ib, platelet Gp IIb, platelet Gp IIIa, von Willebrand factor, or platelet thrombospondin. Species cross-reactivity: Does not recognize mouse GMP-140. Other species have not been tested. Test specimen type: Human Plasma, culture supernatants, cell extracts, or other biological fluids. Sample volume per well: 100 µl 2 8 C Kit Components MK426 UV Plate 1 plate (96 wells) GPDH substrate 1 11 ml Enzyme extraction buffer 1 11 ml Dilution buffer 2 11 ml Performance Characteristics Assay duration: 2 10 min. (following sample addition) Sample volume per well: 25 µl 20 C CELL BIOLOGY 3 Enzyme Immunoassay Kits 137

139 3CELL BIOLOGY Enzyme Immunoassay Kits Rat Heme Oxygenase-1 EIA KIt Rat Heme Oxygenase-1 EIA Kit Cat.# MK assays Application Quantitative Measurement of Rat Heme Oxygenase-1 Heme oxygenase is a microsomal enzyme that degrades heme proteins (e.g. hemoglobin) into the bile pigment biliverdin (which is converted to bilirubin), carbon monoxide and reduced iron (Fe 2+ ). Bilirubin has an anti-inflammatory effect in the body through its strong radical scavenging activity, whereas carbon monoxide has a vasodilative effect on organ blood flow. Heme oxygenase exists as at least two isozymes (heme oxygenase-1 and heme oxygenase-2), which differ in their tissue expression and inducibility. Heme oxygenase-1 (HO-1) is an enzyme whose expression is induced intracellularly in response to various types of stress, and monitoring of HO-1 may prove useful for identifying stress induction. The Rat Heme Oxygenase-1 EIA Kit is a 96-well format in vitro enzyme immunoassay kit for quantitative determination of rat heme oxygenase-1 in rat blood, cell culture supernatants, and various organs. It is a solid phase sandwich EIA that utilizes two mouse monoclonal rat HO-1 antibodies (one of which is coated on the plate, and the other is POD-labeled) for detection of rat HO-1 using a two step incubation method. In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled HO-1 antibody. The reaction between POD and the substrate (H 2 O 2, TMBZ) results in a color development. The amount of sample HO-1 is determined by measuring absorbance using an EIA plate reader. Accurate HO-1 sample concentrations can be determined by comparing sample absorbance values with the absorbance obtained for the Standard plotted on a standard curve. Kit Components MK124 Antibody-coated microtiter plate 1 plate (8 wells 12 strips) Antibody-POD conjugate (lyophilized) 1 vial (for 1 11 ml) Standard (lyophilized) 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Substrate solution 1 vial (1 12 ml) Extraction buffer 1 vial (1 11 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: ng/ml Assay duration: 2.5 hours Specificity: Rat HO-1. Does not recognize rat HO-2. Species cross-reactivity: Does not recognize human, rabbit, guinea pig, or mouse HO-1. Test specimen type: Rat serum, organ extracts or cell culture supernatant Sample volume per well: 100 µl 2 8 C Mouse Heme Oxygenase-1 EIA Kit (Precoated) Mouse Heme Oxygenase-1 EIA Kit Cat.# MK assays Application Quantitative Determination of Mouse Heme Oxygenase-1 in Mouse Cell Culture Supernatants, Mouse Serum and Mouse Organ Extracts Heme oxygenase is known to exist as at least two isozymes (heme oxygenase-1 and heme oxygenase-2). While heme oxygenase-2 is a constitutive enzyme, heme oxygenase-1 (HO-1) is an enzyme whose expression is induced intracellularly in response to various type of stress. The Mouse Heme Oxygenase-1 EIA Kit is a sandwich ELISA kit that uses 2 types of antibodies that recognize mouse heme oxygenase-1 and provides a convenient and highly sensitive method for detecting heme oxygenase-1 expressed in response to stress induction. Monoclonal Antibodies This kit uses clone GTS-1 (Cat.# M174) as the solid antibody and polyclonal Anti- Heme oxygenase-1 as the labeled antibody. Kit Components MK125 Antibody-coated microtiter plate 1 plate (8 wells 12 strips) Antibody-Biotin conjugate (lyophilized) 1 vial (for 1 11 ml) Standard 1 vial (for 1 1 ml) Sample diluent 2 vials (2 11 ml) Avidin-POD conjugate 1 vial (for 1 11 ml) Substrate solution 1 vial (1 12 ml) Extraction buffer 1 vial (1 11 ml) Performance Characteristics Range of assay: ng/ml Assay sensitivity: ng/ml Assay duration: 2.5 hours Specificity: Specific for mouse HO-1 no detectable cross-reaction with mouse HO-2. Does not recognize human, rabbit or guinea pig. Does recognize rat HO-1 with rat HO-1 standard. Test specimen type: Mouse serum, organ extracts, cell culture supernatant. Specimen volume required: If each test sample is run in duplicate, approximately 200 µl. : 2 8 C 138

140 Universal Tyrosine Kinase Assay Kit Universal Tyrosine Kinase Assay Kit Cat.# MK assays Quantitative Measurement of Tyrosine Kinase Activity Analysis of PTK Activity Regulation In vitro Screening of PTK Inhibitors Protein Tyrosine Kinase (PTK) is an important enzyme of the signal transduction pathway which controls cell proliferation, differentiation and carcinogenesis. PTK is classified into two groups: 1) membrane receptor PTKs (e.g., insulin receptor and EGF receptor); and, 2) non-receptor linked PTKs (e.g. Src and FAK). Receptor PTKs contain three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular and transmembrane domains bind to ligands such as hormones and growth factors, while the intracellular domain carries the PTK active site. Receptor PTKs transduce signals directly into the cell by phosphorylation of tyrosine when ligands bind to their domains. Non-receptor linked PTKs lack the transmembrane domain and are divided into three types based upon their location in the cell (i.e., membrane bound, cytoplasma or nucleus). Non-receptor linked PTKs participate in various functions, including transduction and cell-cell communication. The Universal Tyrosine Kinase Assay Kit is an in vitro enzyme immunoassay kit for quantitative determination of tyrosine kinase activity using either adherent or suspension cell culture extracts, and is useful for analyzing regulation of PTK activity and for in vitro screening of PTK inhibitors. The kit utilizes a PTK substrate which is coated onto a 96-well microtiter plate, a phosphotyrosine (PY20)-horse radish peroxidase (HRP) antibody, and HRP substrate (TMBZ). Serial dilutions of a prepared cell extract are added with an ATP solution to the PTK substrate-coated microtiter plate, allowing phosphorylation of tyrosine. The sample solution is then removed, and the plate well washed and blocked. Following blocking, anti-phosphotyrosine (PY20)- HRP is added, incubated and then replaced by the HRP substrate solution (TMBZ). Color development ensues, and the amount of sample PTK activity is determined colorimetrically by comparing the sample s specific absorbance at 450 nm with the absorbance obtained for the Standard plotted on a standard curve. Peptide substrate PTK ATP Phosphorylation of tyrosine P POD Anti phosphorylate tyrosine antibody-pod POD TMBZ Principle of the Universal Tyrosine Assay Kit P Absorbance at 450 nm POD P TMBZ color Kit Components MK410 PTK substrate-coated microplate 1 plate (8 wells 12 strips) Kinase reacting solution 1 vial (1 11 ml) 40 mm ATP-2Na (lyophilized) 2 vials (for ml) Extraction buffer 1 vial (1 11 ml) PTK control (lyophilized) 1 vial (for ml) Anti-phosphotyrosine (PY20)-HRP (lyophilized) 1 vial (for ml) Blocking solution 1 vial (1 11 ml) HRP substrate solution (TMBZ) 1 vial (1 12 ml) Performance Characteristics Range of assay: U/µL Assay sensitivity: U/µL sample (= 32 fmol/well) (as Src kinase) Assay duration: hours Specificity: g-phosphate residue transfer from ATP to substrate. Does not recognize phosphorylated serine or threonine. Test specimen type: Suspension or adherent cell extracts Sample volume per well: 40 µl 2 8 C Application Example 1. Changes in PTK activity with embryo genesis PTK activity levels in Mouse embryos at day 11 and 15 of gestation (full term is 20 days) were measured using the Universal Tyrosine Kinase Assay Kit. Mouse embryo 11 days of gestation 15 days of gestation PTK activity (units/mg protein) Influence of IFN-γ on U937 cells PTK activity level in U937 cells after treatment with IFN-γ (1,000 units/ml) for 48 hours was compared to treated cells. Treatment No treatment With IFN-γ treatment PTK activity ( 10-5 units/10 6 cells) Specific measurements of PTK activities of EGF-R and FAK following immunoprecipitation Cell A431 A431 A431 WiDr WiDr Treatment EGF EGF EGF No treatment No treatment Antibody (quantity/tube) Anti-EGFR* (24 µg) Anti-cSrc** (10 µl) Normal mouse Ig (11 µl) Anti-FAK*** (10 µg) Normal rabbit Ig (10 µl) PTK activity (units/100 6 cells) Not detected 79 Not detected CELL BIOLOGY 3 Enzyme Immunoassay Kits * Cat.# M059 (Mouse monoclonal antibody) ** Chemicon Code: AB1420 (Rabbit antiserum) *** Cat.# M135 (Rabbit antibody) 139

141 3CELL BIOLOGY Albumin EIA Kit (Bovine) Albumin EIA Kit (Bovine) Cat.# MK reactions To Detect Bovine Albumin in Purified Protein Preparations Monitor Albumin Levels in Bovine Organ Extracts or Urine Samples The Bovine Albumin EIA kit uses a bovine albumin-specific monoclonal antibody. It can be used to rapidly detect residual bovine albumin in purified target proteins produced by cultured cells and to monitor albumin levels in bovine organ extracts or urine samples. Bovine albumin purified from serum is widely used as a model protein or a carrier protein in biochemical, molecular biological, and immunological experiments. Other applications include its use as a stabilizer during manufacturing of recombinant biologics and as a nutrient source in animal cell culture. Kit Components MK131 Antibody Coated Microtiter plate 1 plate (8 wells 12 strips) Antibody - POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (2 ml) Substrate Solution 1 vial (12 ml) Performance Characteristics Range of assay: ng/ml Sensitivity: 2.5 ng/ml Assay duration: 2.5 hours for assay protocol Specificity: Specifically measures bovine albumin. Test specimen type: Bovine plasma, serum, urine, organ extracts and cell extracts When assaying bovine serum samples, the recommended dilution is fold or greater. Specimen volume required: 100 µl/assay well. 4 C Enzyme Immunoassay Kits Human Albumin EIA Kit Human Albumin EIA Kit Cat.# MK wells Application Quantitative Determination of Human Albumin in Human Serum and Body Fluid Samples This quantification kit using a human albumin-specific monoclonal antibody can be utilized as a simple tool to monitor albumin levels in human serum and body fluids, among other purposes. Albumin is a protein of about 66 kda present in blood at a very high concentration. In addition to maintaining blood osmotic pressure, it is also involved in transporting fatty acids and a variety of other substances by forming complexes with them. Albumin, which is hepatically synthesized and renally removed, serves as an indicator of liver function. Kit Components MK411 Antibody Coated Microtiter plate 1 plate (8 wells 12 strips) Antibody - POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (2 ml) Substrate Solution 1 vial (12 ml) Performance Characteristics Range of assay: ng/ml Sensitivity: 2.5 ng/ml Assay duration: 2.5 hours for assay protocol Specificity: Specifically measures human albumin Test specimen type: Human plasma, serum, urine, organ extracts and cell extracts. When assaying human serum samples, the recommended dilution is fold or greater. Specimen volume required: 100 µl/assay well 4 C 140

142 Rat IgE EIA Kit Rat IgE EIA Kit Cat.# MK reactions Application Assay IgE in Rat Animal Model studies of Allergic Diseases The Rat IgE EIA Kit is designed to assay rat IgE using two different monoclonal antibodies that are rat IgE-specific and do not cross-react with other rat immunoglobulins. This highly sensitive immunoassay allows accurate determination of the total IgE sera in rat serum or other samples with excellent reproducibility. Immunoglobulins (Ig) are a type of serum globulin proteins, and are involved in the immune process. There are five classes of immunoglobulins (IgG, IgA, IgM, IgD, and IgE) IgE is responsible for allergic responses. Serum IgE may be assayed to identify responses to a panel of allergy-causing substances (allergens). Furthermore, the total amount of IgE in blood is one of the criteria to determine type I allergy diseases and atopic diseases. While mouse is a commonly used experimental animal model in studies of allergic diseases, the usefulness of mouse models is limited by small sample volumes of serum and difficulty in collecting time courses by repeated sampling of the same animal. As a result, rat models have been used increasingly. Performance Characteristics Range of assay: ng/ml Sensitivity: ng/ml Assay duration: 2.5 hours for assay protocol Specificity: Specifically measures Rat IgG Test specimen type: Rat plasma and serum. When assaying rat serum samples, the recommended dilution is 3- to 10-fold. Specimen volume required: 100 µl/assay well is required. 4 C CELL BIOLOGY 3 Enzyme Immunoassay Kits 141

143 3CELL BIOLOGY Rat IgG EIA Kit Rat IgG EIA Kit Cat.# MK assays Assay Rat Immunoglobulin in Serum Measure Culture Supernatants of Hybridomas for Rat Monoclonal Antibody Concentration Features: Measure IgG in Rat Blood Samples and Monitor Antibody Production:in rat-monoclonal antibody-producing hybridomas. High Sensitivity Sandwich ELISA: supplied with a ready-to-use plate coated with solid-phase capture antibody. Perform the Entire Assay in just 60 Minutes The Rat IgG EIA kit contains a rat IgG-specific polyclonal antibody, and is applicable not only for measuring IgG in all rat blood samples in any strain of rat, but also for highly sensitive monitoring of IgG production in rat-monoclonal antibody-producing hybridomas. This kit comes with a ready-to-use plate coated with solid-phase capture antibody. It allows simple detection of rat IgG with good sensitivity and reproducibility in only 60 minutes. Performance Characteristics Range of assay: Standard protocol: ng/ml High-sensitivity protocol: ng/ml Sensitivity: Standard protocol: 10 ng/ml High-sensitivity protocol: 2.5 ng/ml Assay duration: Standard protocol: 3.5 hours High-sensitivity protocol: 1 hour Specificity: Specifically measures Rat IgG Test specimen type: Rat serum and plasma, or Rat hybridoma culture supernatant and ascites or other appropriate biological samples Specimen volume required: 100 µl/assay well Kit Component MK138 Antibody Coated Microtiter plate Antibody - POD Conjugate (lyophilized) Standard (lyophilized) 4 C (96 wells: 8 wells 12 strips) 2 plates for 2 11 ml Enzyme Immunoassay Kits Mouse IgG EIA Kit Mouse IgG EIA Kit Cat.# MK assays Assay Mouse Immunoglobulin in Serum Measure Culture Supernatants of Hybridoma for Mouse Monoclonal Antibody Concentration Features Measure IgG in Mouse Blood, Monitor Antibody Production of Hybridoma Cells, or Test the Quality of Drugs or Research Reagents that use a Monoclonal Antibody High Sensitivity Sandwich ELISA: supplied with a ready-to-use plate coated with solid-phase capture antibody Perform the Entire Assay in just 60 Minutes. Mouse IgG EIA Kit is a quantification kit that uses a mouse IgG-specific polyclonal antibody. It can be used to measure IgG in mouse blood, test the quality of drugs or research reagents that use a monoclonal antibody, and monitor antibody production of hybridoma cells. This kit comes with a ready-to-use plate coated with solid-phase capture antibody. It allows the detection of mouse immunoglobulin with good sensitivity and reproducibility within a short time only 60 minutes for the entire assay. Immunoglobulins (Ig) are antibody proteins that play a key role in antibodymediated immunity. Performance Characteristics Range of assay: Standard protocol: ng/ml High-sensitivity protocol : ng/ml Sensitivity: Standard protocol: 10 ng/ml High-sensitivity protocol: 5 ng/ml Assay duration: Standard protocol: 3.5 hours High-sensitivity protocol: 1 hour Specificity: Specifically measures Mouse IgG Test specimen type: Mouse serum and plasma, mouse hybridoma culture supernatant and ascites, or other appropriate mouse biological samples Specimen volume required: 100 µl/assay well Kit Components MK137 Antibody Coated Microtiter plate Antibody - POD Conjugate (lyophilized) Standard (lyophilized) Sample Diluent Substrate Solution (TMBZ) 4 C (96 wells: 8 wells 12 strips) 2 plates for 2 11 ml for 2 1 ml 60 ml for 2 12 ml 142

144 Human IgG EIA Kit Human IgG EIA Kit Cat.# MK assays Kit Components Antibody Coated Microtiterplate 1 plate (8 wells 12 strips) Antibody - POD Conjugate 1 vial (for 11 ml) Standard 1 vial (for 1 ml) Sample Diluent 2 vials (for 2 11 ml) Substrate Solution 1 vial (12 ml) Performance Characteristics Range of assay: ng/ml Sensitivity: 10.0 ng/ml Assay duration: 2.5 hours Specificity: Specifically measures Human IgG Test specimen type: Human blood and body fluids. When human blood sample is used, the recommended dilution is >10 5 fold as a standard. Specimen volume required: 100 µl/assay well γ-globulins are plasma proteins known as immunoglobulins (Ig) for their roles in the immune response. There are five types of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. IgG is produced by plasma cells differentiated from activated B lymphocytes and functions in many roles: as various types of immune antibodies, as neutralizing antibodies, and as opsonizing antibodies that help capture bacteria. IgG is produced by lymph nodes, spleen, bone marrow, thymus, small intestinal mucosa, and respiratory tract mucosa, among other tissues. Since IgG is produced in response to bacteria, viruses, drugs, and tissue antigens, the blood level of IgG is used as an indication of antigen stimulation. The presence of high IgG levels has been linked to polyclonal hypergammaglobulinemia, collagen disease, asymptomatic M-proteinemia, chronic infections, myeloma, and IgG multiple myeloma, and other pathological conditions and diseases. A low IgG level, on the other hand, is associated with disorders such as agammaglobulinemia or hypogammaglobulinemia, severe immunodeficiency, and nephrosis syndrome. Human IgG EIA uses a human IgG-specific monoclonal antibody. The kit can be used to monitor IgG levels in human blood and biological samples. 4 C CELL BIOLOGY 3 Influenza Virus Typing Set Influenza Virus Typing Kit Cat.# MK assays Application Typing of Influenza Virus A and B Subtypes Influenza viruses are classified into three groups (A, B and C) based upon differences in their nuclear proteins (NP), and are further classified into subtypes according to their surface glycoproteins, neuraminidase (NA) and haemagglutinin (HA). The most prevalent types of influenza virus are influenza A H1, influenza A H3, and influenza B. The influenza A HA protein consists of a head region which contains a target cell receptor domain and a stem region that contains a special amino acid sequence required for fusion of the virus and the target cell. The HA head region, in particular, has especially strong antigenicity and, thus, most anti-ha antibodies recognize this region. However, mutation readily occurs in this region, and typing is used to assess the effect of a vaccination. Furthermore, rapid typing is important due to the short infectious period of this virus. The Influenza Virus Typing Set allows typing of influenza virus A and B subtypes. The Set contains several biotinylated antibodies (two kinds of monoclonal antibodies that distinguish between influenza A subtypes, one monoclonal antibody that binds all types of influenza A viruses, one polyclonal antibody that binds all types of A and B viruses) and Avidin-POD. The sensitivity of this system is equivalent to that of the Peroxidase-anti peroxidase (PAP) method. Set Components MK421 Biotinylated-C179 Antibody µl Biotinylated-F49 Antibody µl Biotinylated-C111 Antibody µl Biotinylated-9D6 Antibody µl Biotinylated-Anti Influenza A, B µl Avidin-POD µl Diluent (for antibody, 4) ml Performance Characteristics Assay sensitivity: Equivalent to PAP method Assay duration: 1.5 hours Specificity: Influenza virus A and B subtypes Test specimen type: MDCK cells infected with the sample virus Antibodies Supplied with this Kit Biotinylated-C179: Human Influenza A Monoclonal antibody (H1N1,H2N2) (Cat.# M145) This antibody specifically recognizes the conformational epitope in the stem region of HA in which amino acid sequences are highly conserved in H1N1 and H2N2 subtypes of influenza A virus. Biotinylated-F49: Human Influenza A Monoclonal antibody (H3N2) (Cat.# M146) This antibody specifically recognizes the HA stem region of the H3N2 influenza A virus subtype. Biotinylated-C111: Human Influenza A Monoclonal antibody (H1, H2, H3) (Cat.# M147) This antibody specifically recognizes the matrix protein in H1N1, H2N2 and H3N2 all subtypes of influenza A virus. Biotinylated-9D6: Human Influenza B Monoclonal antibody (Cat.# M148) This antibody specifically recognizes the influenza B virus nuclear protein (NP). Biotinylated-anti influenza A, B: Polyclonal antibody to human influenza A, B (Cat.# M149) This antibody was created by using a vaccine mixture of influenza A/ Peking/262/95 (H1N1), A/Wuhan/359/95 (H3N2), B/Mie/1/93 and B/ Guandong/5/94 for an immunogen. This antibody reacts equally with both influenza A and B virus. 20 C Enzyme Immunoassay Kits 143

145 3CELL BIOLOGY Apoptosis and Cytotoxicity ApopLadder Ex (Apoptotic DNA Fragment Extraction Kit) ApopLadder Ex Cat.# MK reactions Application Sensitive, Specific and Rapid Method for Isolating Fragmented DNA Apoptosis is associated with the fragmentation of chromosomal DNA into approximately 180 bp nucleosome fragments. The ApopLadder Ex Kit selectively extracts the small, fragmented DNA from apoptotic cells and minimizes chromatin contamination. This method supports highly sensitive detection and quantitation of fragmented DNA. Detection of fragmented DNA following isolation can be observed by gel electrophoresis and can be quantitated by fluorescent staining with SYBR Green I (Molecular Probes, Inc). This kit contains the necessary reagents for cell lysis, enzymatic degradation of protein and RNA, precipitation of the DNA fragments, and a 6X loading dye for gel electrophoresis. ApoPrimer Set Application Detection of Human Bcl-2 Gene Family Expression by RT-PCR The Bcl-2 gene family encodes proteins involved in inhibiting apoptosis. The ApoPrimer Set (Bcl-2 family) is composed of primers used to amplify cdna derived from mrna of 7 members of the human Bcl-2 family (mcl-1, bfl-1, bax-α, bcl-2, bak, bik, bcl-x), as well as β-actin (a control). A positive control RNA, including primer sequences of these 7 members of human Bcl-2 family, and the β-actin primer sequence, is also included. This allows the use of β-actin as an internal control for quantification and for comparison between samples. The PCR products generated from the positive control RNA can be distinguished from the target mrna due to differences in the sizes of the amplified products. Note: Human bcl-x Primer Mix amplifies the cdna from both bcl-xl and bcl-xs. Kit Components MK600 Lysis Buffer ml 10% SDS ml Enzyme A Solution ml Enzyme B Solution ml Precipitant ml 6X Loading Dye ml 20 C ApoPrimer Set Cat.# reactions Kit Components 6623 (primer mix - 10 pmol/µl each) Human mcl-1 Primer Mix 40 µl Human bfl-1 Primer Mix 40 µl Human bax-α Primer Mix 40 µl Human bcl-2 Primer Mix 40 µl Human bak Primer Mix 40 µl Human bik Primer Mix 40 µl Human bcl-x Primer Mix 40 µl Human β-actin Primer Mix 40 µl APO Positive Control RNA 1 (10 6 copies/µl) 24 µl 20 C APO Positive Control RNA 1 bak-s bax-α-s mcl-1-s bcl-x-s bak-a bax-α-a bcl-x-a β-actin-a bik-s bcl-2-s bfl-1-s β-actin-s bik-a bcl-2-a bfl-1-a mcl-1-a poly (A) 144

146 In Situ Apoptosis Detection Set In Situ Apoptosis Detection Set Cat.# MK assays Labeling Safe Buffer Cat.# MK rxns TdT Enzyme Cat.# MK502 1 µg Anti-FITC HRP Conjugate Cat.# MK rxns Control Slides Cat.# MK504 1 slide Permeabilisation Buffer Cat.# MK rxns Application Detection of DNA Strand Breaks in Apoptotic Cells using Flow Cytometry or Fluorescence Microscopy Features Fast: allows quick and easy detection Sensitive: allows detection of single cells at the primary stage of apoptosis Specific: apoptotic cells are stained more specifically than necrotic cells Flexible: Tissue sections and fixed cells samples can be used Two detection methods can be used (Fluorescence Microscopy and Light Microscopy) Individual components are available separately Accurate: a control slide is supplied Safe: non-radioactive Apoptosis plays a significant role in cancer, cell growth, proliferation and cellular response to physical factors (i.e. radiation, heat) and chemical factors (i.e., drugs). One hallmark feature of apoptotic cells is fragmentation of chromatin DNA at the nucleosome level (185 bp). This fragmented DNA can be detected histochemically by terminal labeling. This kit is designed to detect fragmented DNA histochemically by terminal labeling. The TUNEL (TdT-mediated dutp nick end labeling) method is effective for measuring DNA fragmentation resulting from apoptotic activation of intracellular endonucleases. Fluorescein labeled nucleotides are incorporated in situ onto the 3 ends of these DNA fragments, allowing histologic detection of individual cells. Principle TUNEL, an acronym for Terminaldeoxynucleotidyl transferase-mediated dutp-biotin nick end labeling, is an effective method for analyzing DNA fragents resulting from the apoptotic activation of intracellular endonucleases. Fluorochrome labeled nucleotides are incorporated in situ onto the ends of these DNA fragments, allowing histologic detection individual cells. Terminaldeoxynucleotidyl Transferase is used to label 3'-OH ends of DNA fragments that are generated during the apoptosis process. The cells undergoing apoptosis are specifically labeled with fluorescein-dutp with high sensitivity, allowing detection by fluorescence microscopy. Since signal can also be detected by a peroxidase-labeled anti-fluorescence antibody, it is also possible to visualize signal using light microscopy. Kit Components MK500 Labeling Safe Buffer µl TdT (Terminal deoxynucleotidyl transferase) Enzyme µl Anti-FITC HRP Conjugate 1.5 ml Control Slides 2 2 slides Permeabilisation Buffer ml 1 Source E. coli carrying a plasmid which encodes terminal deoxynucleotidyl transferase. 2 Control slide is a paraffin-embeded tissue section of rat mammary gland. When used as a positive control slide, deparaffinization of the section. After deparaffinizing and treatment with Proteinase K, follow the detection protocol required. Shipped at 20 C Store components separately: Buffers and Enzyme at 20 C Conjugate at 4 C* Slides at room temperature** * Store at 4 C once it is thawed. ** Store at room temperature after delivery, kit is shipped at 20 C. Type: Direct TUNEL Labeling Assay Used For Sample Types Technique Time Required Detection Method Assays per Kit Detection of DNA strand breaks in apoptotic cells by flow cytometry or microscopy (light of fluorescence). Cells in suspension (permanent cell lines, normal and tumor cells ex vivo), adherent cells, cytospins, cell smears, frozen or paraffin-embedded tissue sections End-labeling of DNA with fluorescein-dutp followed by direct analysis of fluorescent cells. 1 2 hrs. (not including sample preparation, permeabilization, etc.) Fluorescence and light microscopy or flow cytometry 20 assays CELL BIOLOGY 3 Apoptosis and Cytotoxicity 145

147 LDH Cytotoxicity Detection Kit LDH Cytotoxicity Detection Kit Cat.# MK401 2,000 tests 3CELL BIOLOGY Apoptosis and Cytotoxicity Application Measurement of LDH Enzymatic Activity from Damaged Cells Features Safety: no radioactive isotopes are used Accuracy: assay results obtained with this kit strongly correlate to the number of damaged cells High Sensitivity: low cell numbers ( cells/well) Fast: high-throughput compatible-analyze multiple samples Simple Procedure: prelabeling or washing steps; disposal and radiation safety monitoring required. Lactate dehydrogenase (LDH) is a stable, cytoplasmic enzyme present in most cells. In cell culture, damage to (or rupture of) cell cytoplasmic membranes results in the release of LDH from cells. Thus, quantitation of LDH in cell culture supernatants is used to indicate cell death. The LDH Cytotoxicity Detection Kit is a simple, sensitive, nonradioactive 96-well assay based upon the measurement of lactate dehydrogenase (LDH) released by damaged cells into the cell culture supernatant. With this kit, LDH present in the culture supernatant participates in a coupled reaction converting yellow tetrazolium salt (INT) into red formazan product. The amount of enzyme activity, measured as absorbance using a microplate reader at 490/492 nm, correlates to the number of damaged cells in the culture. The entire procedure including cell growth, reaction procedure, and measurement is performed in a single 96-well plate. Principle The culture supernatant is incubated with the reaction mixture supplied in the kit. LDH activity is then measured in an enzymatic test. (See Figure) 1st step. NAD + is reduced to NADH/H + by the LDH-catalyzed conversion of lactate to pyruvate. 2nd step. The catalyst (diaphorase) transfers H/H + of NADH/H + to the tetrazolium salt INT which is reduced to formazan. This leads to color change from pale yellow to red. Kit Components MK401 Catalyst: (diaphorase/ NAD + ; lyophilized) 5 bottles Dye solution: (containing iodotetrazolium chloride (INT) and sodium lactate) 5 45 ml Performance Characteristics Range of assay: cells/well Assay sensitivity: 0.2 cells/well Assay duration: hour Test specimen type: Cell-free culture supernatant (from cells/ well)(serum-free), and cell extracts. Sample volume per well: 200 µl 20 C Lactic acid Formazan (red) LDH NAD + NADH + H + Diaphorase Principle of Measurement of LDH Cytotoxicity Pyruvic acid Tetrazolium (yellow) 146

148 Premix WST-1 Cell Proliferation Assay System Premix WST-1 Cell Proliferation Assay System Cat.# MK400 2,500 tests Measurement of Cell Proliferation Due to Growth Factors, Cytokines, Mitogens and Nutrients Analysis of Cytotoxic and Cytostatic Compounds Evaluation of Factors which Inhibit Cell Growth Features Safe to use: no radioactive isotopes are required. No volatile organic solvent is required for solubilization Accuracy: absorbance strongly correlates to the number of viable cells Sensitivity: more sensitive than using MTT Ease of use: supplied as a ready-to-use, sterile solution Short reaction time. Entire assay performed in one microtiter plate. No need for washing, harvesting or formazan dye solubilization. High through put compatibleusing a multiwell-elisa reader. No isotope disposal or radiation monitoring required. Flexibility: plates can be read then monitored over time for further color development The Premix WST-1 Cell Proliferation Assay System allows fast and easy colorimetric measurement of cell proliferation and viability of cells cultured in 96-well plates. This assay is based on the cleavage of tetrazolium salts (WST-1) to soluble formazan dye by mitochondrial succinate-tetrazolium reductase in viable cells. As a cell population increases, an increase in the amount of reductase present in the culture supernatant results in a concomitant increase in conversion of WST-1 to formazan dye. Thus, the quantity of formazan dye is directly proportional to the number of metabolically active cells in the medium. Formazan dye formed by metabolically active cells can be quantified by measuring its absorbance (440 nm) using a microtiter plate reader. This kit provides a non-radioactive, spectrophotometric method of determining cell proliferation and viability without harvesting or washing steps. The assay can be performed in a single multi-well plate. Premix WST-1 is supplied as a ready-to-use solution containing WST-1 and an electron coupling reagent, diluted in sterile phosphate buffered saline. Premix WST-1 is diluted 1:10 with µl cultured cells/ well. The kit supplies sufficient reagent for 2,500 assays. Principle Tetrazolium salt (WST-1) is cleaved to soluble formazan dye by the succinatetetrazolium reductase (EC ), which exists in mitochondrial respiratory chain and is active only in viable cells. Total activity of this mitochondrial dehydrogenase in a sample rises with the increase of viable cells. As the increase of enzyme activity leads to an increase of the production of formazan dye, the quantity of formazan dye is related directly with the number of metabolically active cells in the medium. The formazan dye formed by metabolically active cell can be quantitated by measuring its absorbance by ELISA reader. The absorbance of formazan dye solution is in direct proportion to the number of viable cells (see figure). This product is also designed for non-radioactive, spectrophotometric quantification of cell growth and viability during proliferation and chemosensitivity screening experiments. I N N NO2 N N SO3Na WST-1 (slightly red) SO3Na NAD + EC-H Cleavage of the Tetrazolium Salt (WST-1) to Formazan Kit Components MK400 PreMix WST-1* 1 bottle 25 ml * The PreMix WST-1 is a ready-to-use solution containing WST-1 and an electron coupling reagent, diluted in sterile phosphate buffered saline. Shipped at 20 C Store at 20 C (protected from light) If a precipitate forms upon thawing, redissolve the precipitate by warming at 37 C for several minutes with gentle shaking. Once thawed, the solution can be stored at 4 C and protected from light for several weeks. For longer storage, store aliquots at 20 C. Do not subject to further freeze-thaw cycles. References 1. Cook, J. A. & Mitchell, J. B. (1989) Anal. Biochem 179, Mosmann, T. (1983) J. Immunol. Methods 65, Carmichael, J. et al. (1987) Cancer Res. 47, Vistica, D. T. et al. (1991) Cancer Res. 51, Scudiero, D. A. et al. (1988) Cancer Res. 48, Weislow, O. S. et al. (1989) J. Natl. Cancer. Inst. 81, Roehm, N. W. et al. (1991) J. Immunol. Methods 142, Cory, A. H. et al. (1991) Cancer Commun. 3, RS NADH (EC = electron coupling reagent, RS = mitochondrial succinatetetrazolium-reductase system) EC I N N NO2 N N H SO3Na SO3Na Formazan (dark red) CELL BIOLOGY 3 Apoptosis and Cytotoxicity 147

149 3CELL BIOLOGY Antibodies Summary of Antibody Clones Clones are produced by fusing the mouse myeloma cell line P3U1 with BALB/C mouse spleen cells after immunization with appropriate immunogen, unless specified otherwise. Each clone is purified from ascites fluid by ammonium sulfate precipitation and anion-exchange chromatography and suspended in 0.15 M PBS (ph 7.4) 1.0% bovine serum albumin, 0.1% sodium azide (2 mg/ml IgG or IgM). Antibodies are shipped lyophilized. Rehydrate by dissolving in distilled water to yield 2 mg Ig/mL stock solution. The lyophilized antibody is stable at 4 C if stored dry. Solutions should be stored in aliquots at 20 C. Repeated freezing and thawing should be avoided. Product Clone Cat.# Isotype ELISA Titer Quantity E-cadherin (Human) HECD-1 M106 IgG 1 1:10, mg SHE78-7 M126 IgG 2a 1:10, mg P-cadherin (Human) NCC-CAD-299 M127 IgG 1 1 :4, mg E-cadherin (Mouse) ECCD-1 M107 IgG 2b (rat) 0.1 mg ECCD-2 M108 IgG 2a (rat) 0.1 mg P-cadherin (Mouse) PCD-1 M109 IgG 2a (rat) 0.1 mg N-cadherin (Chicken) NCD-2 M110 IgG 2a (rat) 0.1 mg N-cadherin (Human) Polyclonal M mg Fibronectin (Human) FN12-8 M002 IgG 1 1:10, mg FN30-8 M010 IgG 1 1:10, mg Vitronectin (Human) VN58-1 M017 IgG 1 1:3, mg Laminin (Human) LN82-13 M020 IgG 1 1:10, mg von Willebrand Factor (Human) VW92-3 M029 IgG 2a 1:10, mg Heme Oxygenase-1 GST-1 M174 IgG 1 1:10, mg GST-3 M175 IgG 1 1:10, mg Platelet GMP-140 (Human; P-Selectin) WGA-1 M062 IgG 1 1:4, mg PL7-6 M063 IgG 1 1:4, mg Platelet GP Ib (Human; CD42b) WGA-3 M064 IgG 1 1:4, mg PL52-4 M065 IgG 1 1:4, mg GUR20-5 M123 IgG 1 1:4, mg Platelet GP IIb/IIIa (Human; CD41a) PT25-2 M122 IgG 1 1:2, mg Procollagen Type I C-Peptide (Human) PC5-5 M011 IgG 1 1:10, mg PC8-7 M012 IgG 1 1:10, mg Rat Collagen Type 2 (Rat) 20G-12E M193 IgG 1 Colli 2B-11 F M192 IgG 2b 1:10, mg Osteocalcin (Bovine) OC4-30 M041 IgG 2a 1:3, mg OCG2 M042 IgG 2 1:4, mg OCG3 M043 IgG 3 1:1, mg OCG4 M044 IgG 1 1:4, mg Osteocalcin (Mouse) Polyclonal M mg Osteocalcin (Human) GluOC4-5 M171 IgG 1 1: mg Osteocalcin (Human) 5-12H M184 IgG 2b 0.1 mg D-8G M185 IgG mg Osteocalcin (Rat) 6-7H M186 IgG mg 9-12H M187 IgG mg 148

150 Summary of Antibody Clones (continued) Product Clone Cat.# Isotype ELISA titer Quantity Osteocalcin (Mouse), Monoclonal R21C-01A M188 IgG 1 1:1, mg Osteonectin/SPARC OSN4-2 M124 IgG 1 1:4, mg ON1-1 M125 IgG 1 1:4, mg Dentin Matrix Protein 1 Polyclonal M mg Calpastatin (Human) CSL1-5 M045 IgG 1 1:4, mg Insulin (Human) IS11-1 M056 IgG 1 1:2, mg Insulin C (Mouse) Polyclonal M mg Bromodeoxyuridine BU6-4 M050 IgG 1 1:2, mg Influenza A (Human) C179 M145 IgG 2a 1:2, mg F49 M146 IgG 1 1:2, mg C111 M147 IgG 2a 1:2, mg Influenza B (Human) 9D6 M148 IgG 1 1:2, mg Influenza A, B (Human) Polyclonal M149 1:2, mg Taq, DNA Polymerase Monoclonal 9002A 250 units Taq, DNA Polymerase Monoclonal 9002B 1000 units GAPDH Polyclonal M mg Glucagon Polyclonal M mg TRACP Polyclonal M mg Cathepsin K Polyclonal M mg Bone ALP (Rat) * Polyclonal M mg Oct4 (Human) * Monoclonal M221 IgG 2b 0.1 mg Lin28 (Human) * Monoclonal M222 IgG 2b 0.1 mg Sox2 (Human) * Monoclonal M223 IgG 2b 0.1 mg Protein S * ProS 7B-8F M200 IgG 1 1:1, mg Bone Specific Alkaline Phosphatase (Rat) * Polyclonal M mg Histone H3 (Mouse) * Monoclonal MA301B IgG ml Monomethyl Histone H3 (Lys4) (Mouse) * Monoclonal MA302B IgG ml Dimethyi Histone H3 (Lys4) (Mouse) * Monoclonai MA303B IgG ml Trimethyl Histone H3 (Lys4) (Mouse) * Monoclonal MA304B IgG ml Acetyl Histone H3 (Lys9) (Mouse) * Monoclonal MA305B IgG ml Monomethyl Histone H3 (Lys9) (Mouse) * Monoclonal MA306B IgG ml Dimethyl Histone H3 (Lys9) (Mouse) * Monoclonal MA307B IgG ml Anti-Trimethyl Histone H3 (Lys9) (Mouse) * Monoclonal MA308B IgG ml Acetyl Histone H3 (Lys27) (Mouse) * Monoclonal MA309B IgG ml Acetyl Histone H3 (Lys9/27) (Mouse) * Monoclonal MA310B IgG ml Phospho Histone H3 (SerlO) (Mouse) * Monoclonal MA312B IgG ml Ago2 (Human) * 1B1 M211 IgG 2a 0.2 mg Ago2 (Human) for Immunoprecipitation * 1B1 M212 IgG 2a 0.1 mg * Indicates new antibody CELL BIOLOGY 3 Antibodies 149

151 3CELL BIOLOGY Antibodies Anti-cadherin Monoclonal Anti-Human E-cadherin (Clone HECD-1) Cat.# M mg Monoclonal Anti-Human E-cadherin (Clone SHE78-7) Cat.# M mg Monoclonal Anti-Mouse E-cadherin (Clone ECCD-1) Cat.# M mg Monoclonal Anti-Mouse E-cadherin (Clone ECCD-2) Cat.# M mg Monoclonal Anti-Human P-cadherin (Clone NCC-CAD-299) Cat.# M mg Monoclonal Anti-Mouse P-cadherin (Clone PCD-1) Cat.# M mg Monoclonal Anti-Chicken N-cadherin (Clone NCD-2) Cat.# M mg Polyclonal Anti-N-Cadherin Cat.# M mg rabbit polyclonal Monoclonal Anti-Human N-cadherin (Clone Ncad 1-1-3) Cat.# M mg HECD-1/SHE78-7: for paraffin-embedded tissue ECCD-1: for inhibition assay Polyclonal Anti-N-cadherin: for paraffin-embedded tissue Cadherins are transmembrane glycoproteins of amino acids that function as Ca 2+ -dependent adhesion receptors and are responsible for tight intercellular connections 1. Members of the cadherin family include: E- (epithelial), P- (placental), and N- (neural) cadherin, which share a common basic structure but show distinct patterns of expression in tissues. The specific binding properties of each member controls selective cell-to-cell adhesion. Regulated expression of the members during development implicates involvement in morphogenesis. Cadherins may also be involved with tumor invasion and metastasis. 1,2 MAb clones originate from relevant hybridoma cells constructed by fusion between splenocytes of immunized hosts and P3-X63-Ag8 myelomas. HECD-1, SHE78-7, and NCC-CAD-299 are purified from ascitic fluids. The other clones are purified from serum-free culture supernatant (see the table below for applications). Form Lyophilized Monoclonal (M106, M126, M107, M108, M109, M127, M110) Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, not containing any preservative) Polyclonal (M142, M143) Dissolve in 200 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Anti-Cadherin Antibody Specification Table Clone Cat.# Reactivity No Reactivity Application Immunogen Immunized Host Subclass Ab titer E-cadherin HECD-1 M106 m, GP g, Q, k, I R, T,l, y m breast tumor cell line MCF-7 BALB/c K K IgG 1 1:10,000 SHE78-7 M126 m k R, T,l, y Soluble m placenta E-cadherin BALB/c K K IgG 2a 1:10,000 ECCD-1 M107 K m, g, I, Q y K teratocarcinoma cell line F9 Wister k k IgG 2b ECCD-2 M108 K, m, Q g, I T,l K teratocarcinoma and mouse liver Donryu k k IgG 2a E-cadherin fragment P-cadherin NCC-CAD-299 M127 m T,l, y m epidermal carcinoma cell line A-431 BALB/c K K IgG 1 1:4,000 PCD-1 M109 K m, I T,l, y K endoderm cell line PSA5-E Donryu k k IgG 2a N-cadherin NCD-2 M110 I, 9 m, K T,l, y m embryonic neural retina Wister k k IgG 2a Polyclonal M142 m, g, K, m E, P-cadherin R, T,l peptide of m N-cadherin( )- e I KLH conjugate m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 150

152 Structure of Cadherin N Specificity defining region Ca 2+ DXD Transmembrane region β α γ C CELL BIOLOGY DXD : Ca 2+ binding sequence α, β, γ : Catenins associated with the carboxy-terminus of cadherin The amino-terminal extracellular portion of the molecule contains sequences that determine the specificity of each cadherin. Calcium is required for cadherin-mediated adhesion and also protects cadherins against proteolysis. Clone ECCD-1, PCD-1, and NCD-2 recognize residues near Glu 16 of murine E-cadherin, Gly(31) of murine P-cadherin, and ser(31) of chicken N-cadherin, respectively. Tissue Distribution of Cadherins (includes transient expression) 3 Early embryo (until neurulation) All cells at preimplantation Embryonic ectoderm Extraembryonic ectoderm Endoderm Extraembryonic ectoderm Notochord Lateral plate mesoderm Visceral endoderm Late embryo (post-neurulation) Most epithelial tissues Sensory ganglion 12 Cephalic neuroepithelium 13 E-cadherin 9 (M.W. 124,000) P-cadherin 7, 9 (M.W. 118,000) Placenta Mesothelium Pigmented retina Basal layer of epidermis and other squamous epithelia Antibodies 9, 10 N-cadherin (M.W. 127,000) Mesoderm Notochord Neural tissues Lens 8 Cardiac and skeletal muscles Nephric primordia Mesothelium Primordial germ cells Not all tissues are listed (For details, see references and citations therein). References 1. Takeichi, M. (1991) Science, 251, Bussemakers, M. J. G. et al. (1992) Cancer Res., 52, Nose, A. et al. (1990) Cell, 61, Shimoyama, Y. et al. (1989) Cancer Res., 49, Yoshida-Noro, C., et al. (1984) Dev. Biol., 101, Shirayoshi, Y. et al. (1986) Cell Struct. Funct., 11, Nose, A. and Takeichi, M. (1986) J. Cell Biol., 103, Hatta, K. and Takeichi, M. (1986) Nature, 320, Takeichi, M. (1988) Development, 102, Hatta, K. et al. (1987) Dev. Biol., 120, Takeichi, M. (1990) Annu. Rev. Biochem., 59, Shimamura, K. et al. (1992) Dev. Biol., 152, Shimamura, K. and Takeichi, M. (1992) Development, 116, Katayama, M. et al. (1994) Int. J. Oncol., 5, Shimoyama, Y. et al. (1989) Cancer Res., 49, Related Products E-cadherin EIA Kit (Precoated) Cat.# MK assays (2-step sandwich-type EIA) 151

153 Monoclonal Anti-Human Platelet Antibodies Monoclonal Anti-Human Platelet GMP-140 (P-selectin/CD62) (Clone WGA-1) Cat.# M mg Monoclonal Anti-Human Platelet GMP-140 (P-selectin/CD62) (Clone PL7-6) Cat.# M mg 3CELL BIOLOGY These clones are raised against activated human platelet GMP-140. Form (for all antibodies) Lyophilized. Dissolve in 50 µl of distilled water with 0.1% sodium azide (final conc.: 2.0 mg/ml) (for all antibodies) Lyophilized antibody is stable at 4 C for up to 2 years. Reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Anti-Human Platelet GMP-140 Antibody Specification Table Specificity (for GMP-140 antibodies) These antibodies with human GMP-140 (P-Selectin). These do not react with reduced antigen. These do not affect platelet aggregation and platelet adhesion. Each clone recognizes a different epitope on the GMP-140 molecule Clone Cat.# Reactivity No Reactivity Immunogen Immunized host Subclass Ab titer WGA-1 M062 m kefq non-activated platelets TlS6 m platelet BALB/c K IgG 1 1: 4,000 PL7-6 M063 m kefq non-activated platelets TlS6 m platelet BALB/c K IgG 1 1: 4,000 Antibodies Monoclonal Anti-Human Procollagen Type I C-Peptide (PIP) Monoclonal Anti-Human Procollagen Type I C-peptide (PIP) (Clone PC5-5) Cat.# M mg Monoclonal Anti-Human Procollagen Type I C-peptide (PIP) (Clone PC8-7) Cat.# M mg Application These Products can be used to Study Collagen Synthesis by Measuring Procollagen Type I C-Peptide Levels in Cell Culture Supernatants. Collagens (types I, II, III, IV and V) are synthesized as precursor molecules called procollagens. These contain propeptide sequences, at both the amino-terminal and the carboxy-terminal ends. The function of these propeptides is to facilitate the winding of procollagen molecules into a triple-helical conformation within the endoplasmic reticulum. The propeptides are cleaved off from the collagen triple helix molecule during its secretion. The triple helix collagens then polymerize into extracellular collagen fibrils. Clones PC5-5 and PC8-7 recognize procollagen type I C-peptide. They are suitable for the detection of non-denatured antigen released in tissue culture media and for monitoring of collagen synthesis. Form Lyophilized Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Lyophilized antibody is stable at 4 C for up to 2 years. Reconstituted antibody solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Related Product Procollagen Type I C-Peptide (PIP) EIA Kit (Precoated) Cat.# MK101 Anti-Human Procollagen Type I C-Peptide Antibody Specification Table Clone Cat.# Reactivity No Reactivity Immunogen Immunized host Subclass Ab titer PC5-5 M011 m g Qh ke RTl m procollagen type I C-peptide BALB/c K IgG 1 1: 10,000 PC8-7 M012 m g Qh ke RTl m procollagen type I C-peptide BALB/c K IgG 1 1: 10,000 m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 152

154 Anti-Rat Collagen type 2, Monoclonal Anti-Rat Collagen type 2, Monoclonal Cat.# M mg Western Blotting Analysis under Reducing or Non-Reducing Conditions Immunocytochemical Staining Source: Clone: Col II 2B-11F Immunogen: Collagen type II Immunized host: C57BL6 mouse Subclass: IgG2b Antibody titer: 1:10,000 Form: Lyophilized Supplied Reconstitution Solution: 0.1% NaN 3 (in water) Reconstitution: Dissolve the lyophilized antibody in 50 µl of Reconstitution Solution. (final concentration: 2.0 mg/ml) Specificity This product specifically reacts with rat collagen type II. This product doesn t react with rat collagen type I and type III. Cross Reactivity: this antibody does not react with mouse collagen type II. It reacts weakly with bovine collagen type II 4 C. The stock solution (2.0 mg/ml) with Reconstitution Solution should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. CELL BIOLOGY 3 Anti-Rat Collagen type II (col II 20G-12E) Anti-Rat Collagen type II (col II 20G-12E) Cat.# M mg Immunohistochemical Detection on Paraffin Embedded Tissue Sections Antigen Retrieval: Proteinase K Treatment Detection of Collagen Type II by Western Blot Analysis under Non- Reducing Condition Features Clone No:. Col II 20G-12E Rat collagen type II monoclonal antibody and antigen Specificity This antibody specifically reacts with rat collagen type II This antibody does not react with rat collagen type I and type III Cross reactivity This antibody reacts with mouse collagen type II This antibody does not react with mouse collagen type I This antibody reacts with bovine collagen type II weakly Subclass: IgG1 Form: Lyophilized : 4 C This product does not contain preservatives. The stock solution (2.0 mg/ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 for up to 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored. Antibodies 153

155 3CELL BIOLOGY Monoclonal Anti-Human Fibronectin Monoclonal Anti-Human Fibronectin (Clone FN12-8) Cat.# M mg Monoclonal Anti-Human Fibronectin (Clone FN30-8) Cat.# M mg Monoclonal Anti-Human Fibronectin (Heparin-binding domain) (Clone FNH3-8) Cat.# M mg These clones were raised against purified human plasma fibronectin. Each recognizes discrete epitopes of fibronectin (see Figure). For antibody Western blots or histology, clone FN30-8 works best and is also effective for adhesion blockade studies. Clones FN12-8 and FNH3-8 are unique in that they can be used for studies related to fibrin and heparin interactions, respectively. Form Lyophilized Dissolve in 200 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Heparin Fibrin Collagen Heparin Cell Heparin Fibrin Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to up to 1 year. Reference 1. Katayama, M. et al. (1989) Exp. Cell. Res.,185, Related Products Fibronectin Related Peptide (GRGDSP) Cat.# SP001 1 mg Fibronectin Related Peptide (GRGESP) Cat.# SP002 1 mg Fibronectin EIA Kit (Precoated) Cat.# MK assays SAS RGDS FNH3-8 CS1 CS5 FN8-12 :Binding domains H2N H2N 30 K 40 K 20 K 75 K 35 K 30 K IIICS 60 K S S S S COOH COOH Antibodies FN9-1 FNC4-4 FN21-1 (junction) CS: Connecting segments SAS: Synergistic adhesion site RGDS: Arg-Gly-Asp-Ser (cell adhesion signal) FN30-8 FN12-8 Fibronectin Structures FNH3-8 FN8-12 FN1-1 Heparin Fibrin :Binding domains Anti-Human Fibronectin Antibody Specification Table Clone Cat.# Reactivity No Cross Reactivity Application Domain Specificity Immunogen Immunized host Subclass Ab titer FN12-8 M002 m, g f, e, k l Cell m plasma fibronectin BALB/c K IgG 1 1:10,000 adhesion blockade FN30-8 M010 m g, f, TR l Cell m plasma fibronectin BALB/c K IgG 1 1:10,000 e, k adhesion blockade FNH3-8 M115 m, e g, f, k Tl Type III (H12) in third Heparin m plasma fibronectin BALB/c K IgG 1 1:10,000 m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 154

156 Monoclonal Anti-Human Vitronectin Monoclonal Anti-Human Vitronectin (Clone VN58-1) Cat.# M mg This clone was raised against purified human vitronectin. Clone VN58-1 does not interfere with the cell-binding activity of vitronectin. Form Lyophilized Dissolve in 100 µl of distilled water (final conc.: 2.0 mg/ml containing 0.1% sodium azide.) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Related Products Vitronectin EIA Kit (Precoated) Cat.# MK assays Reference 1. Hayman, E. G. et al. (1982) J. Cell. Biol., 95, CELL BIOLOGY Specificity The epitope of this clone exists in amino acid of the N-terminal region of vitronectin. This antibody does not interfere with vitronectin-mediated adhesion. Anti-Human Vitronectin Antibody Specification Table Clone Cat.# Reactivity No Reactivity Application Immunogen Immunized Host Subclass Ab titer VN58-1 M017 m Vitronectin g antigen l TR m vitronectin BALB/c K IgG 1 1:3,000 3 Polyclonal Anti-Tartrate-Resistant Acid Phosphatase (TRACP) Polyclonal Anti-Tartrate-Resistant Acid Phosphatase (TRACP) Cat.# M mg Application Used for Detection of TRACP This antibody is a rabbit polyclonal antibody raised against a peptide at the C-terminas of rat Tartrate-resistant Acid Phosphatase the peptide was conjugated with KLH. Form Lyophilized Source Rabbit polyclonal antibody raised against the peptide ( ) [TYIEASGKSLFKTRLPRRARP] at the C-terminal of rat Tartrate-resistant Acid Phosphatase (TRACP). The peptide conjugated with KLH. Specificity This antibody recognizes amino acid sequence of rat TRACP ( ) [TYIEASGKSLFKTRLPRRARP]. Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months after adding 0.1% NaN 3 (no preservative is included in the lyophilized antibody), and at 20 C for up to 1 year. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. Antibodies 155

157 Monoclonal Anti-Human Laminin Monoclonal Anti-Human Laminin (Clone LN82-13) Cat.# M mg 3CELL BIOLOGY Form Lyophilized Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Specificity This antibody does not inhibit cell adhesion. Anti-Human Laminin Antibody Specification Table Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Related Products Laminin EIA Kit (precoated) Cat.# MK assays Laminin Related Peptide (LGTIPG) Cat.# SP004 1 mg Laminin Related Peptide (YIGSR) Cat.# SP003 1 mg Clone Cat.# Reactivity No Reactivity Immunogen Immunized Host Subclass Ab titer LN82-13 M020 γ 1 chain of N-terminal region m antigen cross reacts with e K antigen l TR m laminin BALB/c K IgG 1 1:10,000 Monoclonal Anti-Osteonectin (SPARC) Antibodies Monoclonal Anti-Osteonectin/SPARC (Clone OSN4-2) Cat.# M mg Monoclonal Anti-Osteonectin/SPARC (Clone ON1-1) Cat.# M mg Application These Clones can be Used for Detection of Activated Platelet These clones were raised against bone-derived bovine osteonectin (ON1-1), and human platelet derived osteonectin (OSN4-2). The clones react with thrombinstimulated platelets, not with non-stimulated platelets. Form Lyophilized. Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Specificity These products can react with both bone-derived and platelet-derived osteonectin. These products react with thrombin-stimulated platelet (flow cytometry). These clones react with human and bovine osteocalcin with the same efficiency (western blotting). These clones cross react with rabbit and porcine antigen (ELISA). Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Anti-Osteonectin Antibody Specification Table Clone Cat.# Reactivity No Reactivity Immunogen OSN4-2 M124 m g osteonectin cross reacts with ef ON1-1 M125 m g osteonectin cross reacts with ef non-activated platelet non-activated platelet l T6 Elisa l TR6 Elisa Immunized host Subclass Ab titer m platelet osteonectin BALB/c K IgG 1 1:4,000 g bone osteonectin BALB/c K IgG 1 1:4,000 m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 156

158 Polyclonal Anti-Dentin Matrix Protein-1 Polyclonal Anti-Dentin Matrix Protein-1 (DMP-1) Cat.# M mg rabbit polyclonal This product is a rabbit polyclonal antibody raised against an N-terminal region of rat Dentin Matrix Protein-1. Source Rabbit polyclonal antibody raised against a peptide (aa90-111) near the N-terminus of rat Dentin Matrix Protein-1 (DMP-1). The peptide was conjugated with KLH. Form Lyophilized. Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Avoid repeated freeze-thaw cycles. For long-term storage of lyophilized antibodies, store at 20 C. CELL BIOLOGY Polyclonal Anti-Dentin Matrix Protein-1 Antibody Specification Table Clone Cat.# Reactivity Immunogen DMP-1 M176 m K k DMP-1(90-111) SGDDTFGDEDNGPGPEEQWGG R l N-terminal of k Dentin Matrix Protein- 1(DMP-1) conjugated with KLH Immunized host Subclass Ab titer eserum IgG 2-5 µg for immunoblotting 3 Monoclonal Anti-Heme Oxygenase-1 Monoclonal Anti-Heme Oxygenase-1 (Clone GTS-1) Cat.# M mg Monoclonal Anti-Heme Oxygenase-1 (Clone GTS-3) Cat.# M mg Monoclonal Anti-Heme Oxygenase-1 Peroxidase-labeled (Clone GTS-1) Cat.# M µl These Products are Sodium Azide Free Can be used for Inhibiting the Heme Oxygenase-1 These antibodies react with heme oxygenase-1. Form M174, M175 Lyophilized. Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, not containing any preservative) Specificity These antibodies do not cross-react with heme oxygenase-2. These antibodies inhibit the activity of human or rat oxygenase-1. Clone GTS-1 reacts with human, mouse and rat heme oxygenase-1. Clone GTS-3 reacts with human and rat heme oxygenase-1. M174, M175: Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution should be stored in aliquots at 20 C or should be stored at 4 C for up to 6 months after sodium azide is added (final conc. 0.1%) as a preservative by the user. Note: Do not add NaN 3 as a preservative since it inhibits the reactions. Related Product Rat Heme Oxygenase-1 EIA Kit (Precoated) Cat.# MK assays Antibodies Monoclonal Anti-Heme Oxygenase-1 Antibody Specification Table Clone Cat.# Reactivity No Reactivity Immunogen Immunized host Subclass Ab titer GTS-1 M174 m kk e, heme oxygenase-2 l RT k heme oxygenase-1 BALB/c K IgG 1 1:10,000 GTS-3 M175 m k e, heme oxygenase-2 l RT k heme oxygenase-1 BALB/c K IgG 1 1:10,

159 3CELL BIOLOGY Antibodies Monoclonal Anti-Bovine Osteocalcin Monoclonal Anti-Bovine Osteocalcin (Clone OC4-30) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG2) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG3) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG4) Cat.# M mg These clones were raised against bovine osteocalcin, and equally recognize both the bovine and the human osteocalcins. Clone OC4-30 is specific for γ-carboxylated osteocalcin. Form Lyophilized Dissolve in 50 µl of distilled water with 0.1% sodium azide (final conc. of antibody: 2.0 mg/ml) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Anti-Bovine Osteocalcin Antibody Specification Table Clone Cat.# Reactivity No Reactivity OC4-30 M041 g m ke Q f K with GIG decarboxylated osteocalcin Related Products Gla-Type Osteocalcin (Gla-OC) EIA Kit (Precoated) Cat.# MK assays Undercarboxylated Osteocalcin (Glu-OC) EIA Kit (Precoated) Cat.# MK assays References 1. Hauschka, P.U., Lian, J.B., Gallop, P.M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, Engvall, E. and Perlmann, P. (1972) J. Immunol. 109, Kayama, N., Ohara, K., Yokota, H., Kurome, T., Katayama, M., Hino, F., Kato, I. and Akai, T. (1991) J. Immunol. Methods 139, Vergnaud, P. et al. (1997) J. Clin. Endocrinol. Metab., 82, Immunogen Immunized host l RT g osteocalcin BALB/c K includes g-carboxylated residue at position 17 Epitope Subclass Ab titer IgG 2a 1:3,000 OCG2 M042 g m K k l RT g osteocalcin BALB/c K residue IgG 1 1:4,000 OCG3 M043 gkefggm K l R(weaker)T g osteocalcin BALB/c K residue IgG 3 1:1,000 OCG4 M044 g m QeG f GI K k l RT g osteocalcin BALB/c K residue 4-9 IgG 1 1:4,000 Monoclonal Anti-Bovine Osteocalcin Monoclonal Anti-Rat Osteocalcin(Clone D-8G) Cat.# M mg Monoclonal Anti-Rat Osteocalcin(Clone 6-7H) Cat.# M mg Monoclonal Anti-Rat Osteocalcin(Clone 9-12H) Cat.# M mg Form Lyophilized Dissolve in 50 µl of distilled water with 0.1% sodium azide (final conc. of antibody: 2.0 mg/ml) Anti-Rat Osteocalcin Antibody Specification Table Clone Cat.# Reactivity No Reactivity D-8G M185 k g f G G and donkey Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Immunogen Immunized host Subclass Ab titer m R k osteocalcin BALB/c K IgG 1 1:1, H M186 g m Rl k osteocalcin BALB/c K IgG 1 1:1, H M187 k m g l k osteocalcin BALB/c K IgG 1 1:1,000 m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 158

160 Monoclonal Anti-Human Undercarboxylated Osteocalcin and Osteocalcin Monoclonal Anti-Human Undercarboxylated Osteocalcin (Clone GluOC4-5) Cat.# M mg Monoclonal Anti-Human Osteocalcin (5-12H) Cat.# M mg M171. This clone was raised against human osteocalcin peptide. It specifically recognizes human osteocalcin with decarboxylated glutamic acid residues, and does not recognize Gla-type osteocalcin. M184. This monoclonal antibody was obtained by fusing the mouse myeloma cell-line P3U1 with spleen cells of C57BL6 mouse after immunization with KLHconjugated peptide from human osteocalcin sequence (amino acids 1-6 YLYQWL). The monoclonal antibody was harvested from scid mouse ascitic fluid. Form Lyophilized. Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, containing 0.1% sodium azide) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Specificity M171 specifically reacts with human osteocalcin with glutamic acid residues (decarboxylated) at positions 21 and 24. The epitope recognized by M184 is amino acid residues 1 6 of human osteocalcin. It does not react with bovine rat, guinea pig, porcine, pigeon or goose osteocalcin. Related Products Polyclonal Anti-Mouse Osteocalcin (Clone moc (1-20)) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OC4-30) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG2) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG3) Cat.# M mg Monoclonal Anti-Bovine Osteocalcin (Clone OCG4) Cat.# M mg Gla-Type Osteocalcin(Gla-OC) EIA Kit (Precoated) Cat.# MK assays Undercarboxylated Osteocalcin (Glu-OC) EIA Kit Cat.# MK assays (Precoated) Reference 1. Vergnaud, P. et al. (1997) J. Clin. Endocrinol. Metab. 82, CELL BIOLOGY 3 Anti-Human Undercarboxylated Osteocalcin Antibody Specification Table Clone Cat.# Reactivity GluOC4-5 M171 m osteocalcin w/glutamic acids residues Xreacts with gekqgfg GP GP No Reactivity Gla-type osteocalcin Rl Immunogen peptide of m osteocalcin(14-30)- KLH conjugate Immunized host Subclass Ab titer BALB/c K IgG 1 1:500 Antibodies Anti-Mouse Osteocalcin Polyclonal Anti-Mouse Osteocalcin ( Clone moc(1-20) ) Cat.# M mg Monoclonal Anti-Mouse Osteocalcin(Clone R21C-01A) Cat.# M mg This product was raised against N-terminal peptide of mouse osteocalcin, and specifically recognizes mouse osteocalcin. It does not cross react with rat osteocalcin. Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Form Lyophilized. Dissolve in 50 µl of distilled water containing 0.1% Sodium azide (final antibody conc.: 2.0 mg/ml. Specificity This product recognizes intercellular N-terminal amino acid sequence of mouse osteocalcin (1-20) [YLGAS VPSPD PLEPT REQCE] Polyclonal Anti-Mouse Osteocalcin Antibody Specification Table Clone Cat.# Reactivity No Reactivity Application Immunogen Immunized Host moc(1-20) Polyclonal R21C-01A Monoclonal M173 K Osteocalcin No cross reactivity with k Osteocalcin R N-terminal peptide of K osteocalcin-klh conjugate e M188 K Osteocalcin g k m Rl peptide of K osteocalcin-klh conjugate SDk 159

161 3CELL BIOLOGY Monoclonal Anti-von Willebrand Factor Monoclonal Anti-Human von Willebrand Factor (vwf) (Clone VW92-3) Cat.# M mg Application Clone can be used to Study the Inhibition of von Willebrand Factor by the Vitronectin Receptor. Clone VW92-3 was obtained using a V8 Protease III fragment of human plasma von Willebrand factor as immunogen. This antibody specifically recognizes human von Willebrand factor and does not cross react with bovine von Willebrand factor. Form Lyophilized. Provided with reconstitution solution (0.1% NaN 3 in water) Anti-von Willebrand Factor Antibody Specification Table Clone Cat.# Reactivity No Reactivity Immunogen VW92-3 M029 m g T l Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored 4 C for up to 6 months, and at 20 C for up to 1 year. References 1. Fujimura,Y., Titani, K., Holland, L.Z., Russell, S.R., Roberts, J.R., Elder, J.H., Ruggeri, Z.M. and Zimmerman, T.S. (1986) J. Biol. Chem., 261, Girma, J.P., Chopek, M.W., Titani, K. and Davie, E.W. (1986) Biochemistry, 25, Fujimura, Y., Miyata, S., Nishida, S., Miura, S., Kaneda, M., Yoshida, A., Fukui, H., Katayama, M., Tuddenham, E.G.D., Usami, Y. and Titani, K. (1992) Thromb. Haemostasis, 68, 464. m plasma von Willebrand factor Immunized Host Subclass Ab titer BALB/c K IgG 2b 1:10,000 Antibodies Fr. III (170 kda x 2) VW53-2 VW92-3 Different epitopes N S S S S N N S S S S N N S S S S N in vivo Proteolysis in vivo Proteolysis V8 V8 V8 Fr. I (50 kda) VW33-5 S S S S S S C C C C C C Fr. I (50 kda) VW33-5 Fr. II (110 kda x 2) Fr.: fragment **vw factor is a polymeric protein which is digested by V8 protease into 3 domains. VW28-1 VW40-1 VW1-2 Different epitopes Structure of von Willebrand Factor and the Location of Epitopes Recognized by Takara s Antibodies m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 160

162 Monoclonal Anti-Human Calpastatin Monoclonal Anti-Human Calpastatin (Clone CSL1-5) Cat.# M mg Application Studies on Domain Structures of Calpastatin, or Ca 2+ -Dependent Enzymes by Measurement of Calpastatin in Supernatant of Cell Cultures This monoclonal anti-human calpastatin antibody (CSL1-5 clone) was raised against recombinant muscle-type calpastatin. Form Lyophilized. Dissolve in 50 µl of distilled water 0.1% sodium azide (final conc.: 2.0 mg/ml) Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Related Product Calpastatin Related Peptide (TYIEELGKREVTIPPKYR) Cat.# SP007 1 mg References 1. Asada, K., Ishino, Y., Shimada, M., Shimojo, T., Endo, M., Kimizuka., F., Kato, I., Maki, M., Hatanaka, M. and Murachi, T. (1989) J. Enzyme Inhib. 3, Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, Engvall, E. and Perlmann, P. (1972) J. Immunol. 109, Uemori, T. et al. (1990) Biochem. Biophys. Res. Comm. 166, Yokota, H. et al. (1991) Mol. Cell. Probes 5, CELL BIOLOGY N-terminus antibody C-terminus 3 Antibodies Recognition Sites of the Anti-Human Calpastatin Antibodies Anti-Human Calpastatin Factor Antibody Specification Table Clone Cat.# Reacts with Not React with Immunogen Immunized host Subclass Antibody titer CSL1-5 M045 m l Tl m calpastatin BALB/c K IgG 1 1:4,000 Monoclonal Anti-Bromodeoxyuridine Monoclonal Anti-Bromodeoxyuridine (Clone BU6-4) Cat.# M mg Application Studies on Cell Growth Activity by Detecting the Amount of Bromodeoxyuridine Incorporated into DNA Form Lyophilized. Dissolve in 50 µl of distilled water with 0.1% sodium azide (final conc.: 2.0 mg/ml) Anti-Bromodeoxyuridine Antibody Specification Table Clone Cat.# Reactivity No Reactivity M050 Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Immunogen Immunized host Subclass Antibody titer BU6-4 M050 bromodeoxyuridine l 6 bromodeoxyuridine-bsa conjugate BALB/c K IgG 1 1:2,

163 3CELL BIOLOGY Monoclonal Anti-Human and Mouse Insulin Monoclonal Anti-Human Insulin (Clone IS11-1) Cat.# M mg Anti-Mouse Insulin, Polyclonal Cat.# M mg Anti-Human Insulin C, Polyclonal Cat.# M mg Application Analytical Studies on the Structure, Function and Metabolism of Insulin These antibodies are suitable for studies on insulin. Anti-Human and Mouse Insulin Antibody Specification Table Form Lyophilized. Dissolve in 100 µl of distilled water with 0.1% sodium azide (final conc.: 2.0 mg/ml) M178 and M056 Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months, and at 20 C for up to 1 year. Clone Cat.# Reactivity No Reactivity Immunogen Immunized host Subclass Antibody titer IS11-1 M056 m gf l T m insulin BALB/c K IgG 1 1:2,000 Polyclonal M178 K amino acid sequence [SPGDLQTLALEVAR] human insulin C RT Intercellular amino acid peptide of mouse insulin (71-84) [SPGDLQTLALEVAR] GP Antibodies Ago2 (Human), Monoclonal (Clone 1B1) Anti-Human Ago2, Monoclonal (Clone 1B1) Cat.# M mg Anti-Human Ago2, Monoclonal for IP Cat.# M mg Monoclonal antibody generated against the N-terminal region of human recombinant protein Ago2, a member of the Argonaute family. The Ago2 protein is a transcription factor that is required for RNA-mediated gene silencing (RNAi). Argonaute proteins bind small interfering RNA (sirna) fragments and have endonuclease activity against mrna that is complementary to the bound sirna fragment. Form Lyophilized. M211 and M212 4 C. This product does not contain preservatives. The stock solution (2.0 mg/ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored. Ago2 (Human), Monoclonal (Clone 1B1) Antibody Specification Table Clone Cat.# Reactivity No Reactivity Immunogen Immunized host Subclass Antibody titer 1B1 M211 m l mago2 BALB/c K IgG 2a 1B1 M212 m S mago2 BALB/c K IgG 2a 162

164 Anti-Human Oct4, Monoclonal Anti-Human Oct4, Monoclonal Cat.# M mg Detection of Human Oct4 by Western Blot Immunochemical Staining Features: Specificity: This product specifically reacts with human Oct4 Subclass: IgG 2b Clone No.: OCT413-12F Form: Lyophilized Reconstitute the lyophilized antibody in 50 μl of distilled water (final antibody concentration 2.0 mg/ml). See product data sheet for dilution instructions. This monocolnal antibody was raised by fusing the P341 mouse myeloma cell line with lymph node cells of C47B46 mouse with recombinant human Oct4 protein. The MAb reacts specifically with human Oct4. 4 C The lyophilized antibody does not contain preservatives. The stock solution (2.0 mg/ ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored. CELL BIOLOGY Anti-Human Lin28, Monoclonal Anti-Human Lin28, Monoclonal Cat.# M mg Detection of Human Lin28 by Western Blot Immunochemical Staining This monoconal antibody was raised by fusing the P3Ul mouse myeloma cell line with lymph node cells of C57B46 mouse with human Lin28 recombinant protein. The antibody specifically recognizes human Lin28. 3 Features: Specificity: This product specifically reacts with human Lin28 Subclass: IgG 2b Clone No.: LIN A Form: Lyophilized Reconstitute the lyophilized antibody in 50 μl of distilled water (final concentration 2.0 mg/ml). Add NaN 3 to final conc. of 0.1% if stock solution is to be stored at 4 C. See product data sheet for dilution instructions. 4 C The lyophilized antibody does not contain a preservative. The stock solution (2.0 mg/ml) should be stored in aliquots at 20 C for 1 year, or should be stored at 4 C for 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored. Note: Be sure to store the antibody at a minimum concentration of 2.0 mg/ml. A lower antibody concentration may result in decreased stability. Antibodies Anti-Human Sox2, Monoclonal Anti-Human Sox2, Monoclonal Cat.# M mg Detection of Human Sox2 by Western Blot Immunocytochemical Staining Features: Specificity: This product specifically reacts with human Sox2 Subclass: IgG 2b Clone No.: SOX 2 9B-12H Form: Lyophilized Reconstitute the lyophilized antibody in 50 μl of distilled water (final concentration 2.0 mg/ml). Add NaN 3 to final conc. of 0.1% if stock solution is to be stored at 4 C. See product data sheet for dilution instructions. This product was raised against the KLH-conjugated peptide from human Sox2 sequence (amino acid : GSPTYSMSYSQQGTPGMA). The antibody specifically reacts with human Sox2. 4 C The lyophilized antibody does not contain preservatives. The stock solution (2.0 mg/ ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored. Note: Be sure to store the antibody at a minimum concentration of 2.0 mg/ml. A lower antibody concentration may result in decreased stability. m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 163

165 Polyclonal Anti-GAPDH Polyclonal Anti-GAPDH Cat.# M mg 3CELL BIOLOGY Antibodies Detection of GAPDH by Western Blotting Analysis Detection of GAPDH by Flow Cytometry Analysis This polyclonal antibody was raised in rabbit using chicken GAPDH as immunogen. It can be used to detect GAPDH by western blot under reducing or non reducing conditions and by flow cytometry. Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution (2.0 mg/ml) should be stored in aliquots at 20 C for up to 1 year, or at 4 C for up to 6 months. Avoid repeated freeze-thaw cycles. This diluted antibody should not be stored. Polyclonal Anti-Glucagon (Guinea Pig) Source Rabbit polyclonal antibody raised against the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of chicken. Form Lyophilized Specificity This antibody recognizes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of chicken. It cross reacts with human, monkey, porcine, bovine and mouse GAPDH. Polyclonal Anti-Glucagon Cat.# M mg Application Used for Detection of Glucagon This antibody was raised in guinea pig against a peptide of human glucagon conjugated with KLH. It can be used for immunohistochemical detection of glucagon or paraffin-embedded or frozen tissue sections. Form Lyophilized Specificity This product specifically recognizes amino acid sequence of human Glucagaon [HSQGTFTSDYSKYLDSRRAQDFVQWLMNT]. Lyophilized antibody is stable at 4 C for up to 2 years. The reconstituted solution can be stored at 4 C for up to 6 months or in aliquots at 20 C for up to 1 year. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. Anti-Human Cathepsin K Anti-Human Cathepsin K Cat.# M mg Detection of Human Cathepsin K by Western Blotting Analysis under reducing and non-reducing Conditions (2 5 µg/ml) Immunohistochemical Detection on Paraffin Embedded and Frozen Tissue without Antigen Retrieval (5 30 µg/ml) Source Polyclonal Immunogen: The peptide ( ) [CWAFSSVGALEGQLKKKTG] of human Cathepsin K conjugated with KLH Immunized host: rabbit Kit Components Supplied Reconstitution Solution 0.1% NaN 3 in water Reconstitution: Dissolve the lyophilized antibody in 50 µl of Reconstitution Solution. (final antibody concentration 2.0 mg/ml) Store lypophilized antibody at 4 C. The stock solution (2.0 mg/ml) prepared with Reconstitution Solution should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. Form Lyophilized Specificity This antibody recognizes amino acid sequence of human Cathepsin K ( ) [CWAFSSVGALEGQLKKKTG] This antibody specifically reacts with human Cathepsin K Cross Reactivity: the antibody does not react with mouse or rat cathepsin K m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 164

166 Influenza Virus (Human) Monoclonal Anti-Human Influenza A (H1N1, H2N2) (Clone C179) Cat.# M mg Monoclonal Anti-Human Influenza A (H3N2) (Clone F49) Cat.# M mg Monoclonal Anti-Human Influenza A (H1, H2, H3) (Clone C111) Cat.# M mg Monoclonal Anti-Human Influenza B (Clone 9D6) Cat.# M mg Polyclonal Anti-Human Influenza A, B (rabbit polyclonal) Cat.# M mg Clones C179, F49, C111, 9D6 can be used for Influenza Virus Detection and Typing The Polyclonal Antibody can be used for Detection of both Influenza A and B Virus Clones C179, F49, C111, 9D6 and the Polyclonal Antibody can be used for immunohistochemical Staining by the PAP (peroxidadeantiperoxidase) Staining Method Clone C179 can be used for Neutralization Activity Test of Influenza Virus H1N1 and H2N2 Clone C179 can be used in Experiments to Protect Animals Against Influenza Virus Infection Clone C111, 9D6 can be used for Western Blotting Analysis Influenza virus is classified into three groups (A, B, and C). Clinically prevalent types of Influenza virus are H1, H3 and B strain. The structure of Influenza virus is shown on the next page. Influenza virus is further classified into subtypes based on variances in Neuraminidase (NA) and Haemagglutinin (HA) that are the surface glycoprotein of the virus. H1N1 subtype and H3N2 subtype are known to cause severe febrile illness. An influenza virus particle consists of a head region and a stem region. The head region has a receptor domain and the stem region contains a region required for fusion between the virus and the target cell. Takara offers several influenza antibodies that are useful for various viral detection and typing methods, as well as neutralization assays. These antibodies are for research use only. Works with Swine Flu Form Lyophilized Monoclonal (Cat.# M145, M146, M147, M148) Dissolve in 50 µl of distilled water (final conc.: 2.0 mg/ml, not containing any preservative) Polyclonal (Cat.# M149) Dissolve in 200 µl of distilled water (final conc.: 2.0 mg/ml, not containing any preservative) Lyophilized antibodies are stable at 4 C for up to 2 years. The reconstituted solution should be stored in aliquots at 20 C. Note: For research use only. Not for use in diagnostic or therapeutic procedures. Clone Immun. C179 Influenza A virus A/ Okuda/57 strain (H2N2) F49 Influenza A virus A2/ Aichi2/68/ strain (H3N2) C111 Influenza A virus A/ Okuda/57 strain (H2N2) 9D6 Influenza B virus B/ Nagasaki/1/87 strain Polyclonal Vaccine mixture of influenza, A/Peking/262/95(H1N1), A/Wuhan/395/95(H3N2), B/Mie/1/93 and B/ Guandong/05/94 Immun. host BALB/c mouse BALB/c mouse BALB/c mouse BALB/c mouse Rabbit Antib Sub-class titer IgG2a 1:2,000 IgG1 1:2,000 IgG2a 1:2,000 IgG1 1:2,000 CELL BIOLOGY 3 Antibodies 165

167 Anti-Human Influenza Virus Antibody Specification Table 3CELL BIOLOGY Antibodies Clone Cat.# Reactivity C179 M145 H5N3 I influenza, H1N1, H2N2 F49 M146 m influenza, H3N2, H4N6, H10N7 C111 M147 H1N1, H2N2, H3N2 9D6 M148 NP of Influenza B Polyclonal M149 H1N1, H3N2 and B/Nagasaki /1/87 Table 1. Titers of Monoclonal Antibodies Against Influenza A and B Viruses Virus type and strain No Reactivity H3N2 C179 F49 C111 Staining Neutralizing Staining Staining (H1N1) A/PR/8/ A/Bangkok/10/ A/Yamagata/120/ A/Osaka/930/ A/Suita/1/ (H2N2) A/Okuda/ A/Adachi/2/ A/Kaizuka/2/ A/Izumi/5/ A/Takatsuki/4/ (H3N2) A/Aichi/2/ A/Fukuoka/C29/ A/Sichuan/2/ A/Ibaraki/1/ A/Suita/1/ (H3N8) A/Budgreiger/Aichi/1/ (H4N6) A/duck/Czechoslovakia/1/ (H5N3) A/whistling swan/shimane/476/ (H5N9) A/turkey/Ontario/7732/ (H6N5) A/shearwater/Australia/1/ (H6N6) A/whistling swan/shimane/37/ (H7N7) A/tufted duck/shimane/124r/80 + (H8N4) A/turkey/Ontario/6118/68 + (H9N2) A/turkey/Wisconsin/ (H10N7) A/chiken/Germany N / (H11N6) A/duck/England/56 + (B) B/Nagasaki/1/87 Influenza virus typing, neutralization of H1N1 and H2N2 Immunogen Influenza A virus A/Okuda/57 strain (H2N2) H1N1, H2N2 Influenza virus typing Influenza A virus A2/Aichi2/68/ strain (H3N2) B virus Influenza A virus l, Influenza virus typing and detection l, Influenza virus typing and detection Detection of influenza A and B virus Influenza A virus A/Okuda/57 strain (H2N2) Table 2. Staining Titers of Monoclonal Antibody Against Influenza B Virus recognized protein 100 nm Immunized host 9D6 NP B/Lee/40 + B/Osaka/2/70 + B/Gihu/2/73 + B/USSR/100/84 + B/Tokyo/676/84 + B/Norway/1/84 + B/Ibaragi/2/85 + B/Victoria/2/78 + B/Nagasaki/1/87 + B/Aichi/5/88 + B/HongKong/22/89 + B/Mie/1/93 + B/Guandong/5/94 + Structure of Influenza Virus Subclass Haemagglutinin (HA) Neuraminidase (NA) Matrix Protein (M1) Nuclear Protein (NP) Antibody titer BALB/c K IgG 2a 1:2,000 BALB/c K IgG 1 1:2,000 BALB/c K IgG 2a 1:2,000 Influenza B virus B/Nagasaki/1/87 strain BALB/c K IgG 1 1:2,000 Vaccine mixture of influenza A/Peking/262/95(H1N1), A/Wuhan/395/95(H3N2), B/Mie/1/93 and B/Guandong/05/94 e m = human g= bovine Q= dog k = rat I = chicken K = mouse e = rabbit f = porcine h = horse G = sheep GP = guinea pig G = goat 9 = xenopus R= Histology on paraffin embedded tissue T= Histology on frozen tissue sections l = Western blotting analysis under reducing or non-reducing conditions y = Inhibition assay dependent cell-cell contact S = Immunoprecipitation 6= flow cytometry l = Western blotting analysis under non-reducing and non-heating condition 166

168 Monoclonal Antibodies to Rat Osteocalcin Monoclonal Antibody to Rat Osteocalcin (D-8G) Cat.# M mg Monoclonal Antibody to Rat Osteocalcin (9-12H) Cat.# M mg Monoclonal Antibody to Rat Osteocalcin (6-7H) Cat.# M mg M185 Immunohistochemical Detection of Osteocalcin on Formalin Fixed Decalcified Paraffin Embedded Tissue. (Antigen Retrieval: Proteinase K treatment) Cannot be used for Western Blotting Analysis M186 Immunohistochemical Detection of Osteocalcin on Formalin Fixed Decalcified Paraffin Embedded Tissue (Antigen Retrieval: Proteinase K treatment) Western Blotting Analysis Reducing or non-reducing conditions M187 Western Blotting Analysis using Rat Bone Extract under reducing or non-reducing conditions Cannot be used for immunohistochemical detection. Source Monoclonal antibody was obtained by fusing the mouse myeloma cell-line P3U1 with spleen cells of BALB/c mouse after immunization with KLH-conjugated peptide from Rat osteocalcin sequence (M185 and M186 amino acids 1-25: YLNNGLGAPAPYPDPLEPHREVCEL; M187 amino acids 38-50: FQDAYKRIYGTTV). The monoclonal antibody was harvested from ascitic fluid of BALBc Mouse. Form Lyophilized Lyophilized antibody is stable at 4 for 2 years.the stock solution (2.0 mg/ml) with Reconstitution Solution should be stored in aliquots at 20 for 1 year, or should be stored at 4 for 6 months. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. CELL BIOLOGY 3 Anti-Mouse Osteocalcin Monoclonal Antibody to Mouse Osteocalcin (R21C-01A) Cat.# M mg This antibody can be used for immunohistochemical detection of osteocalcin on formalin fixed decalcified paraffin embedded mouse tissue. (Antigen retrieval: Proteinase K treatment) This product can be used for western blotting analysis using mouse bone extract under reducing or non-reducing condition : Monoclonal antibody was obtained by fusing the mouse myeloma cell-line P3U1 with spleen cells of SD rat after immunization with KLH-conjugated peptide from mouse osteocalcin sequence (amino acids CDELSDQYGLKTAYKRIYGITI). The monoclonal antibody was harvested from ascites fluid of scid mouse. Specificity Clone Specificity Subclass R21C-01A This product recognizes amino acid sequence of mouse osteocalcin (aa25-46) Rat IgG 2a Form Lyophilized Antibody titer 1:1000 times (ELISA method using a microtiter plate with immobilized peptide antigen) Working concentration: 5-10 μg/ml (for immunohistochemical staining by color detection) 5-10 μg/ml (for Western blotting by color detection) Lyophilized antibody is stable at 4 C for up to 2 years. Reconsulted antibody solution (2.0 mg/ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. Antibodies Anti-Rat Bone Specific Alkaline Phosphatase, Polyclonal Anti-Rat Bone Specific Alkaline Phosphatase, Polyclonal Cat.# M mg Detection of Bone Specific Alkaline Phosphatase by Western blot analysis under non-reducing condition Immunohistochemical Detection on Paraffin Embedded Tissue Section (no need for antigen retrieval ) This product was raised against conjugate of KLH and the peptide (20 49) [PEKEKDPKYWRDQAQETLKYALELQKLNTN] that illustrates an exceptional homology with human and rat Bone Specific Alkaline Phosphatase. Specificity: This antibody specifically reacts with rat Bone Specific Alkaline Phosphatase Cross reactivity: This antibody reacts with mouse antigen This antibody reacts faintly with human antigen Form Lyophilized : 4 C This polyclonal antibody does not contain preservatives. The stock solution (2.0 mg/ ml) should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for 6 months after adding 0.1% sodium azide. Avoid repeated freeze-thaw cycles. Diluted antibody should not be stored

169 3CELL BIOLOGY Antibodies Anti-Protein S, Monoclonal Anti-Protein S, Monoclonal (Clone ProS 7B-8F) Cat.# M mg For detection of ProS2-fused Protein expressed using pcold ProS2 DNA Use for Western Blotting Analysis under reducing or non-reducing condition (0.5-1 µg/ml) Suitable for Immunoprecipitation (10 µg/ml) Use with E. coli cold shock expression system pcold ProS2 DNA (Cat.# 3371) Anti-Protein S is a monoclonal antibody used for detection of ProS2-fused protein expressed by using pcold ProS2 DNA. ProS2 is about 23 kda of a solubilization tag which is a tandem-dimer of the N-terminal domain of Protein S, a soluble protein derived from myxobacteria Myxococcus xanthus. Mouse Section Slide Source: Clone: ProS 7B-8F Immunogen: ProS2 tag protein Immunized Host: C57Bl6 mouse Subclass: IgG 1 Antibody titer: 1:2000 Form Lyophilized Supplied Reconstitution Solution: 0.1% NaN3 (in water) Reconstitution: Dissolve the lyophilized antibody in 50 µl of Reconstitution Solution (final concentration: 2.0 mg/ml). Cross Reativity: This product does not cross react with endogenous proteins derived from E. coli. It does not cross react with TF (Trigger Factor) tag. 4 C. The stock solution (2.0 mg/ml) with Reconstitution Solution should be stored in aliquots at 20 C for up to 1 year, or should be stored at 4 C for up to 6 months. Avoid repeated freeze-thaw cycles. The diluted antibody should not be stored. Mouse Section Slide (newborn, whole-mount) Cat.# MK311 5 Slices Immunohistochemistry Histological analysis Features 8 µm sections mounted on glass slides Tissue was prepared by fixation in formalin for 48 h, dehydrated, and paraffin-embedded This product provides sectioned, glass-mounted tissue that is ready-to-use for immunohistochemical analysis or histological studies. Tissue sections are formalinfixed and paraffin embedded sections. They were prepared from 1-day-old C57BL6 mouse. Anti-Histone Antibodies Features Host: Mouse Antigen: 13 amino acid residues from the N-terminus of human Histone H3.1 Buffer: PBS % sodium azide Immunoprecipitation: 1 5 µg/5 µl Sepharose Immunostaining: µg/ml Immunoblot: µg/ml Source Culture supernatant of serum-free medium Source Mouse C57BL6: (1 day after birth) Form Slice thickness : 8 μm Note: Slide-to-slide variations in organs occur due to inherent variability in serial sectioning. 20 C Bring sections to room temperature just prior to use. Anti-Histone H3, Mouse Monoclonal Antibody Cat.# MA301B 100 µl Anti-Monomethyl Histone H3 (Lys4), Mouse Monoclonal Antibody Cat.# MA302B 100 µl Anti-Dimethyl Histone H3 (Lys4), Mouse Monoclonal Antibody Cat.# MA303B 100 µl Anti-Trimethyl Histone H3 (Lys4), Mouse Monoclonal Antibody Cat.# MA304B 100 µl Anti-Acetyl Histone H3 (Lys9), Mouse Monoclonal Antibody Cat.# MA305B 100 µl Anti-Monomethyl Histone H3 (Lys9), Mouse Monoclonal Antibody Cat.# MA306B 100 µl Anti-Dimethyl Histone H3 (Lys9), Mouse Monoclonal Antibody Cat.# MA307B 100 µl Anti-Trimethyl Histone H3 (Lys9), Mouse Monoclonal Antibody Cat.# MA308B 100 µl Anti-Acetyl Histone H3 (Lys27), Mouse Monoclonal Antibody Cat.# MA309B 100 µl Anti-Acetyl Histone H3 (Lys9/27), Mouse Monoclonal Antibody Cat.# MA310B 100 µl Anti-Phospho Histone H3 (Ser10), Mouse Monoclonal Antibody Cat.# MA312B 100 µl Concentration: 1 mg/ml Notes: Mouse IgG interacts weakly with protein A and protein G. Use for immunoprecipitation. Store below 20 C References: 1. Kimura, H., Hayashi-Takanaka, Y., Goto, Y., Takizawa, N., Nozaki, N. The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies,cell Struct Funct. 2008:33(1):

170 Yatalase Enzyme Yatalase Enzyme Cat.# T017 2 g Application Lyse Cell Walls of Filamentous Fungi Features Excellent Thermostability Can be Stored at Room Temperature Efficiently Digests Native Chitin Can be used Alone to Prepare Protoplasts from Filamentous Fungi Yatalase Enzyme is used to lyse the cell walls of filamentous fungi. The product is prepared from culture supernatants of Corynebacterium sp. OZ-21, and consists mainly of chitinase, chitobiase and b-1,3-glucanase. This product is manufactured by Ozeki Corporation. Source Corynebacterium sp. OZ-21 Form Lyophilized powder (containing lactose) Unit Definition Chitinase activity: One unit of chitinase activity is determined as the amount required to release 1 µmol of N-acetylglucosamine from chitin in 1 minute. Chitobiase activity: One unit of chitobiase activity is defined as the amount of enzyme required to release 1 µmol of p-nitrophenol from p-nitrophenyl-n-acetylb-d-glucosaminide in 1 minute. Lytic activity: One unit of enzyme activity is defined as the amount required to cause a 1% decrease in absorbance in 1 hour. Lyophilized preparation is stable for at least 1 year at 4 C. Specific Activities Chitinase activity: Approximately 50 units/g powder Chitobiase activity: Approximately 500 units/g powder Lytic activity against cell walls: Approximately 10,000 units/g powder Properties Optimal ph: ph 7.2 Optimal temperature for enzyme activity: 37 C Yatalase Enzyme Activities Enzyme Activity Activity (units/g power) Chitinase 50 Chitobiase 500 Chitosanase 19 b-1,3-glucanase 300 Protease 31 Cell-well lysing activity 10,000 Conditions for Protoplast Preparation Strains Culture Media Culture Conditions Conditions for Preparing Protoplasts * Aspergillus oryzae Czapek-Dox + 0.5% casamino acid (ph 5.6) * Aspergillus kawach Czapek-Dox + 0.5% casamino acid (ph 5.6) * Aspergillus terreus * Penicillium citrinum * Penicillium lanosum * Tricoderma koningii * Mucor hiemalis * Rhizopus nigricans * Pleurotus ostreatus * Corpinus cinereus * Lentinus edodes Dextrin-peptone (ph 5.5) 30 C, 20 hours aeration (Rotary, 140 rpm) 30 C, 20 hours Stationary culture 30 C, 20 hours aeration (Rotary, 140 rpm) 2% Malt extract 30 C, 12 hours Stationary culture (Sporangiospores in germ) OSG or MYG medium (ph 5.5) * Monascus sp. Dextrin-peptone (ph 5.5) C, 3-4 days Stationary culture 25 C, 20 hours aeration (Rotary at 140 rpm) Species Yield of protoplasts ( 10 6 /g-wet cells) Aspergillus oryzae 70 Aspergillus kawachi 2 Aspergillus terreus 21 Penicillium citrinum 20 Penicillium lanosum 50 Trichoderma koningii 100 Monascus sp. 14 Mucor hiemalis 8 Rhizopus nigricans 21 Pleurotus ostreatus 11 Coprinus cinereus 20 Lentinus edodes 2 Yields of Protoplasts 2% Yatalase solution 0.6 M (NH 4 ) 2 SO 4 50 mm maleate buffer (ph 5.5) 30 C, Reciprocal shaking (at rpm), 2 3 hours 2% Yatalase solution 0.6 M (NH 4 ) 2 SO 4 50 mm maleate buffer (ph 5.5) 30 C, Reciprocal shaking (at rpm), 2 3 hours 2% Yatalase solution 0.6 M (NH 4 ) 2 SO 4 50 mm maleate buffer (ph 5.5) 30 C, Reciprocal shaking (at rpm), 2 3 hours 2% Yatalase solution 0.5 M MgSO 4 50 mm maleate buffer (ph 5.5) 30 C, Reciprocal shaking (at rpm), 4 hours 2% Yatalase solution 0.6 M MgSO 4 50 mm maleate buffer (ph 5.5) 2% Yatalase solution 0.6 M (NH 4 ) 2 SO 4 50 mm maleate buffer (ph 5.5) 30 C, Reciprocal shaking (at rpm), 2 3 hours CELL BIOLOGY 3 Others 169

171 3CELL BIOLOGY Others TBS (Tris-Buffered Saline) Powder TBS (Tris-Buffered Saline) Powder Cat.# T packages (for 30 liters) Usage TBS is used to dissociate tissues, and to wash and disperse cells. TBS is used to conduct Bound/Free separation and to wash solid-phases. Preparation Dissolve 1 package in distilled water to make up a total volume of 1 liter. (50 mm, ph 7.6) TBE (Tris-Borate-EDTA) Powder Usage TBE is used as a buffer for nucleic acid agarose gel electrophoresis. TBE is used to for automated sequencing protocols. Preparation Dissolve 1 package in distilled water to bring to a total volume of 1 liter. (1X, M, ph ) Components 1 package (for 1 liter) contains: Tris (hydroxymethyl) aminomethane 1.24 g Tris (hydroxymethyl) aminomethane hydrochloride 6.27 g Sodium chloride 8.77 g This product is manufactured by Roman Industries Co., Ltd. Store power in a dry state at room temperature. After rehydration, seal buffer vessel tightly and store at 4 C. References 1. Lam, S. C. T., Plow, E. F., Dsouza, S. E., Cherech, D. A., Frelinder III, A. L., and Ginsberg, M. H. (1989) J. Biol. Chem. 263: Gan, Z. and Wells, W. W. (1988) J. Biol. Chem. 263:9050. TBE (Tris-borate-EDTA) Powder Cat.# T packages (for 30 liters) Components One package (for 1 liter) contains: Tris (hydroxymethyl) aminomethane g Borate 5.51 g EDTA 0.75 g This product is manufactured by Roman Industries Co., Ltd. Store powder in a dry state at room temperature. After rehydration, seal buffer vessel tightly and store at 4 C. Note: This product may solidify when stored in humid conditions. Product quality is not affected

172 Phosphate Buffered Salts (PBS) Phosphate Buffered Salts (PBS) Cat.# T tablets (for 20 liters) Usage PBS is used to dissociate tissues, and to wash and disperse cells. PBS is used to conduct Bound/Free separation and for wash steps during the EIA method. Preparation Dissolve 10 tablets in distilled water and to bring to a total volume of 1 liter. This will yield a 9.75 mm solution of PBS (ph ). Components 10 tablets (for 1 liter) contains: Sodium chloride 8,000 mg Potassium chloride 200 mg Sodium monohydrogen phosphate (anhydrous) 1,150 mg Potassium dihydrogen phosphate (anhydrous) 200 mg This product is manufactured by Roman Industries Co., Ltd. Store tablets at room temperature. After rehydration, seal buffer vessel tightly and store at 4 C. Reference 1. Dulbecco, R. and Vogt, M. (1954) J. Exp. Med. 99:167. CELL BIOLOGY Tris-Glycine-SDS Powder Tris-Glycine-SDS Powder Cat.# T packages (for 25 liters) Usage This product is used as a buffer for SDS-polyacrylamide gel electrophoresis. Preparation Dissolve 1 package in distilled water and bring to a total volume of 500 ml. This will yield a 50 mm solution at ph 8.3. Components One package (for 500 ml) contains: Tris (hydroxymethyl) aminomethane 3.0 g Glycine 14.4 g Sodium dodecyl sulfate 0.5 g This product is manufactured by Roman Industries Co., Ltd. Store powder at room temperature. After rehydration, seal buffer vessel tightly and store at 4 C. References 1. Laemmli, U. K. (1970) Nature 227: Suzuki, K. (1977) Genetics 31:43. 3 Others Tris-Glycine Powder Tris-Glycine Powder Cat.# T packages (for 25 liters) Usage This product is used as a buffer for polyacrylamide gel electrophoresis. Preparation Dissolve 1 package in distilled water and bring to a total volume of 500 ml. This will yield a 50 mm solution at ph 8.3. Components One package (for 500 ml) contains: Tris (hydroxymethyl) aminomethane 3.0 g Glycine 14.4 g This product is manufactured by Roman Industries Co., Ltd. Store dry at room temperature. After hydration, seal tightly and store at 4 C. Reference 1. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121:

173 3CELL BIOLOGY Others Cartilage Staining Kit Cartilage Staining Kit Cat.# MK reactions Application Detection of Aggrecan and Type II Collagen in Cartilage. Features Includes 3 Stains and 1 Antibody: for use in cartilage studies Toluidine Blue and Safranin-O are Cationic Stains (basic dyes): that stain acidic proteoglycan present in cartilage tissues Fast Green is the Contrast Stain of Safranin-O: and strongly stains noncollagen sites Kit contains an Anti-Rat type II Collagen Monoclonal Antibody: to detect type II collagen of rat chondrocyte and cartilage tissue by immunostaining This kit can be used to easily detect aggrecan and type II collagen in cartilage. It includes three stains: Toluidine blue, Safranin-O and Fast Green. Toluidine blue and Safranin-O are cationic stains (basic dyes) that stain acidic proteoglycan present in cartilage tissues. Toluidine blue, also called metachromasia dye, shows subtle color changes depending on the tertiary structure of the sample. Cytoplasm stains light blue, nuclear regions dark blue, and mast cell purple. The main constituents of articular cartilage include aggrecan and type II collagen, both of which are proteolyzed when cartilage degenerates. Aggrecan, the major proteoglycan in cartilage, is a macromolecule consisting of an approximately 210- kda core protein bound to a large number of sulfated chondroitin-side chains and multiple sulfated keratin-side chains. The core protein also has a domain capable of Adipoinducer Reagent (for animal cell) binding to hyaluronate produced by synoviocytes. Type II collagen is a fibrillary element responsible for maintaining the special mesh structure in healthy cartilage and forms the structure that takes into its pores waterretentive proteoglycan. Type II collagen, unlike types I and III, exhibits a foreignbody antigenicity. Anti-type II collagen antibody, once generated in the body, induces rheumatism or arthritis. Safranin-O, which binds to glucosaminoglucan and shows an orange color, is often used to stain articular cartilage. Fast green, the contrast stain of Safranin-O, is a sulfate-group containing acidic substrate, which binds strongly to the amino group on protein and thereby strongly stains the noncollagen sites. This kit also contains an Anti-Rat type II Collagen monoclonal Antibody to detect type II collagen of rat chondrocyte and cartilage tissue by immunostaining. Kit Components (1) Fixation solution 30 ml (Citrate buffer (ph5.4) containing 45% Acetone and 10% methanol) (2) 1% Acetic acid 10 ml (3) 0.1% Safranin O 10 ml (4) 0.1% Fast green 10 ml (5) 0.1% Toluidine blue (ph 4.1) 10 ml (6) Anti-Rat Collagen type II monoclonal Antibody 100 µl (7) Diluent for Antibody 10 ml 20 C Adipoinducer Reagent (for animal cell) Cat.# MK429 1 kit Features Contains three of the most frequently used Adipocyte Differentiation- Inducers: insulin, 3-isobutyl-1-methylxanthine and dexamethasone Efficiently induces Adipocyte Differentiation in Mouse Embryonic Fibroblast 3T3-L1 cells (ATCC No. CL-173), rat brown/white preadipocytes, mouse/rat/rabbit bone marrow cells and other animal cells 20 C This reagent efficiently induces adipocyte differentiation in mouse embryonic fibroblast 3T3-L1 cells (ATCC No. CL-173), rat brown/white preadipocytes, mouse/rat/ rabbit bone marrow cells and other animal cells. This kit contains three of the most frequently used adipocyte differentiation-inducers: insulin, 3-isobutyl-1-methylxanthine and dexamethasone. Adipose differentiation can be achieved by culturing cells in an appropriate medium containing these inducer reagents. Adipocyte differentiation is an area of scientific focus because adipose tissue plays an important role in energy homeostasis. Researchers are studying transcriptional regulation of adipocyte differentiation and the role epigenetic regulation may play in differentiation. 3T3-L1 cells are one of the models used to study this process because they differentiate efficiently into adipocytes when induced by insulin, 3-isobutyl-1-methylxanthine and dexamethasone. Peptide Coating Kit Peptide Coating Kit Cat.# MK reactions 5 plates Detection of Anti-Peptide Antibody (e.g. ELISA) Detection of Peptide Antigen (e.g. competitive ELISA) Generally, low molecular weight peptides and synthetic peptides do not absorb spontaneously to 96 well microtiter plates. This kit is designed to allow optimum coating of peptides on plates effectively and easily. The Peptide Coating Kit includes buffer, 96 well microplates and blocking solution specialized for subsequent ELISA. Principle The free carboxyl group of the peptide to be used for coating should be covalently linked with EDC (Coupling Reagent), and subsequently linked with an amino group that is attached to the surface of the microplate through a peptide bond. As a result, the peptide will be directly conjugated to the surface of the microplates through a peptide bond. Kit Components MK100 Reaction Plate 96 well (8 well 12 strips) 5 plates Coupling Reagent 1 vial (for 1 5 ml) Reaction Buffer 1 vial (1 50 ml) Blocking Solution 2 vials (2 50 ml) 2 8 C 172

174 overview Protein Research Products & Recombinant Protein Folding & Expression Chaperone Plasmid Set Chaperone Competent Cells pcold DNA pcold TF DNA pcold ProS2 DNA SPP System (Single Protein Production System) mrna Interferase -MazF Brevibacillus Expression System II Human Cell Free Protein Expression System Bacillus Subtilus Secretory Protein Expression System Protein Research N-Terminal Deblocking & Analysis Pfu Pyroglutamate Aminopeptidase Pfu Methionine Aminopeptidase Pfu Aminopeptidase I Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) Protein Fragmentation Arginylendopeptidase Asparaginylendopeptidase Endoproteinase Asp-N Pfu Protease S Protease inhibition Calpastatin PROTEIN RESEARCH 4 In vitro refolding Chaperonin GroE Corystein (Purothionin) Reagent Protein Disulfide-Isomerase Protein Folding Products Takara s Protein Folding and Expression Products Protein Expression Products Table of Contents Maximize solubility and active protein production Chaperonin GroE Corystein Chaperone Plasmid Set Expression Vectors Protein pcold Vectors pcold TF Vectors Increase protein purity and yield Optimize refolding conditions of recombinant proteins Refolding CA Kit Chaperonin GroE Corystein SPP System (single protein production) pmazf Structural Analysis (NMR) Active Soluble Protein ready for purification 173

175 4PROTEIN RESEARCH Overview Chaperone Plasmid Set Chaperone Plasmid Set Cat.# Set Application Facilitates Correct in vivo Folding of Expressed Recombinant Proteins expressed in E. coli The Chaperone Plasmid Set consists of 5 different plasmids, each of which is designed to express multiple molecular chaperones that function as a chaperone team to enable protein folding. Co-expression of a target protein with one of these chaperone teams increases the recovery of soluble proteins. Each plasmid carries an origin of replication derived from pacyc and a Cm r gene, which allows use with E. coli expression systems utilizing ColE1-type plasmids containing an ampicillin resistance gene as a marker. The chaperone genes are situated downstream of an arab or Pzt-1 (tet) promoter. Therefore, expression of target proteins and chaperones can be induced individually if the target gene is placed under the control of other promoters (e.g. lac). These plasmids also contain the necessary regulator (arac or tet r ) for each promoter. Use this set to perform a two step method to construct target/chaperone co-expression systems. In the first step prepare an E. coli host transformed with only the chaperone plasmid. The 2nd step is to prepare competent cells from this new strain and transform this strain with a plasmid expressing the target protein. Compatible E.coli expression systems These chaperone plasmids carry the pacyc origin of replication and a chloramphenicol resistance gene (Cm r gene); this allows use with standard E. coli expression systems utilizing ColE1-type plasmids with ampicillin resistance gene as a marker. This system cannot be used in combination with chloramphenicolresistant E. coli host strains or expression plasmids that carry the chloramphenicol-resistance gene. For example, E. coli BL21(DE3), which is often used with pet systems etc., is acceptable as a host strain, but E. coli BL21(DE3) plyss and BL21(DE3) plyse, which contains the plyss or plyse plasmids which have the pacyc replication origin and the Cm r gene, cannot be used with this system. Set Components 3340 Plasmid pg-kje8 (10 ng/µl) 100 µl Plasmid pgro7 (10 ng/µl) 100 µl Plasmid pkje7 (10 ng/µl) 100 µl Plasmid pgtf2 (10 ng/µl) 100 µl Plasmid ptf16 (10 ng/µl) 100 µl No. Plasmid Chaperone Promoter Inducer Resistant Marker References 1 pg-kje8 dnak-dnaj-grpe arab L-Arabinose groes-groel Pzt1 Tetracyclin Cm 2, 3 2 pgro7 groes-groel arab L-araBinose Cm 2 3 pkje7 dnak-dnaj-grpe arab L-Arabinose Cm 2 4 pg-tf2 groes-groel-tig Pzt1 Tetracyclin Cm 3 5 ptf16 tig arab L-Arabinose Cm 3 arac arab dnak Cm r pkje7 7.2 kb pacyc ori dnaj Cm r arac arab dnak dnaj pacyc ori grpe grpe pg-kje kb groel rrnbt1t2 groes Pzt1 tetr groes pacyc ori Cm r groel pg-tf2 8.3 kb tetr Pzt1 Cm r pacyc ori arac pgro7 5.4 kb arab groes groel tig Cm r arac arab ptf16 5 kb pacyc ori tig Maps of Takara s Chaperone Plasmids Notice The intellectual property of the plasmids supplied in this product is owned by TAKARA BIO INC. This product is intended for research purposes only. If you would like to request sequence information and/or permission to modify plasmids supplied in Takara's Chaperone Plasmid Set, you must first request the Chaperone Plasmid Set Infomation Request Form. See product information at: for more information. Possible Model for Chaperone-Assisted Protein Folding in E. coli 174

176 Chaperone Competent Cells Chaperone Competent Cells BL21 Set Cat.# x 6 species x 100 µl Chaperone Competent Cell pg-kje8 BL21 Cat.# x 100 µl Chaperone Competent Cell pgro7/bl21 Cat.# x 100 µl Chaperone Competent Cell pkje7/bl21 Cat.# x 100 µl Chaperone Competent Cell pg-tf26/bl21 Cat.# x 100 µl Chaperone Competent Cell ptf16/bl21 Cat.# x 100 µl Chaperone Competent Cell BL21 Cat.# x 100 µl Convenient and Correct in vivo Folding of Expressed Proteins in E. coli Molecular chaperones are extensively-studied proteins involved in the in vivo protein folding process. E. coli BL21 is an E. coli strain derived from E. coli B which possesses defects in the lon and ompt Outer membrane proteases. E. coli BL21 is commonly used for recombinant protein expression because it generates highly stable expressed protein. Takara s Chaperone Competent Cells are competent Escerichia coli strain BL21 cells containing one of Takara s five Chaperone Plasmids. Takara s Chaperone Plasmids (pg-kje8, pgro7, pkje7, pg-tf2, ptf16) were developed by the HSP Research Institute, Inc., and are designed for efficient expression of chaperone teams - molecular chaperones which work cooperatively in the cell. Coexpression of a target protein with one of these chaperone teams increases soluble recovery the target, and may facilitate expression of proteins which are often unrecoverable by conventional expression methods due to inclusion body formation. Coexpression of a target protein and chaperone team normally requires three steps: 1. Transformation of host E. coli with a chaperone plasmid; 2. Prepare competent cells using these transformants and, 3. Transform the cells with a plasmid expressing the target protein. The Chaperone Competent Cells only require one transformation to obtain E. coli coexpressing a target protein and chaperone team. In addition, TaKaRa s Competent BL21 cell are available as a control. These cells can be used for protein expression with Takara s pcold DNA series. They cannot be used with the T7 promoter-based expression, such as the pet system, because the BL21 strain does not express T7 RNA Polymerase. Set Component Chaperone Competent Cells BL21 Set (Cat.# 9120) Chaperone Competent Cell pg-kje8/bl21 Chaperone Competent Cell pgro7/bl21 Chaperone Competent Cell pkje7/bl21 Chaperone Competent Cell pg-tf2/bl21 Chaperone Competent Cell ptf16/bl21 TaKaRa Competent Cell BL21 puc19 DNA (0.1 ng/μl) SOC medium* μl μl μl μl μl μl 1 10 μl 20 1 ml Each Cat.# contains the following components Competent Cell μl puc19 DNA (0.1 ng/μl) 1 10 μl SOC medium* 10 1 ml * SOC medium: 2% Tryptone, 0.5% Yeast extract, 10 mm NaCl, 2.5 mm KCl, 10 mm MgSO4, 10 mm MgCl2, 20 mm Glucose 80 C Related Products pcold I DNA μg pcold II DNA μg pcold III DNA μg pcold IV DNA μg pcold Vector Set Set (ea. 5 μg) Chaperone Plasmid Set Set References 1) Thomas, J.G., et al. (1997) Appl. Biochem. Biotech.,66, ) Nishihara, K., et al. (1998), Appl. Environ. Microbiol.,64, ) Nishihara, K., et al. (2000), Appl. Environ. Microbiol.,66, Limited Use Label License: Cat.# 9120:[M7][M8]; Cat.# 9121, 9122, 9124 and 9125:[M7] PROTEIN RESEARCH 4 Recombinant Protein Folding & Expression 175

177 4PROTEIN RESEARCH Recombinant Protein Folding & Expression Cold Shock Expression System pcold DNA pcold Vector Set Cat.# Set (ea. 5 µg) pcold I DNA Cat.# µg pcold II DNA Cat.# µg pcold III DNA Cat.# µg pcold IV DNA Cat.# µg Protein Expression utilizing the Cold Shock Promoter (cspa). Features High Yield Recombinant Protein: up to 60% of expressed intracellular protein consists of target protein Soluble Expression is Increased: proteins that are insoluble in conventional expression systems can be expressed in soluble form Wide Range of E.coli Hosts: compatible with most E.coli strains Can be Combined with Chaperone Plasmids Vectors: when used in conjuction with one of Takara s Chaperone Plasmids, the amount of recoverable soluble protein can be further increased Radioisotope Labeling: up to 90% of newly expressed cellular protein is labeled target protein Takara s pcold Expression Vectors offer cold shock expression technology for high purity, high yield protein production. The series includes four different vectors that utilize the cold shock Protein A (cspa) promoter to direct expression of recombinant protein in E. coli. These vectors selectively induce target protein synthesis at low temperatures (15 C) when the synthesis of other proteins is suppressed and protease activity is decreased. This results in high yield of the target protein (60% of intracellular protein). In addition to the cspa promoter, all four vectors contain a lac operator (for control of expression), ampicillin resistance gene (amp r ), ColE1 origin of replication, M13 IG fragment, and multiple cloning site (MCS). Furthermore, three of the vectors also contain either a translation enhancing element (TEE), His-Taq sequence, and/or Factor Xa cleavage site. These vectors work equally well for synthesis of non-labeled and radiolabeled proteins and can be used in conjunction with Takara s Chaperone Plasmid Set (Cat.# 3340). Note: Licensing agreement is required. Please see our website at takara. This product is for research use only. It must not be used for clinical purposes or in vitro diagnosis. GenBank Accession Nos. Accession No. pcold I AB pcold II AB pcold III AB pcold IV AB Form 10 mm Tris-HCl (ph 8.0), 1 mm EDTA Chain Length pcold I DNA : pcold II DNA : pcold III DNA : pcold IV DNA : 4,407 bp 4,392 bp 4,377 bp 4,359 bp Features of Takara s pcold Vectors Translation Factor Xa Enhancing His Tag Cleavage Site Element pcold I DNA Ο Ο Ο pcold II DNA Ο Ο pcold III DNA Ο pcold IV DNA Purity Agaraose Gel Electrophoresis confirms that it contains over 70% doublestranded covalently closed circular DNA (RF I). Maintainence of cloning sites confirmed. Single site cleavage by restriction enzymes Nde I, Sac I, Kpn I, Xho I, BamH I, EcoR I, Hind I, Sal I, Pst I and Xba I confirmed. 20 C pcold Vector Map 176

178 Application: Using the pcold Expression Vectors In the following examples, genes that showed poor expression levels or solubility in the T7 promoter expression system were expressed in the cold-shock expression system. pcold I DNA was used as an expression vector and BL21 was used as host for expression. Expression from T7 promoter-driven vectors was conducted using the standard protocol with IPTG used to induce expression and culturing at 37 C. kda N.C T7 pcold kda T7 pcold T S T S T: Entire protein fraction S: Soluble fraction Expression level increased PROTEIN RESEARCH 31 Expression enabled CBB staining of the entire protein fraction CBB staining 4 Figure 1. Expression of Human Gene A. Human gene A (~ 31 kda) was expressed in both the T7 system and the cold-shock expression system. No expression was observed in the T7 system, but target gene was expressed using the cold-shock expression system. kda T7 pcold T S T S CBB staining T: Entire protein fraction S: Soluble fraction Expression level increased Figure 2. Expression of Thermophilic Gene B. Increased protein expression and improved solubility were observed when expressing thermophilic gene B (~ 30 kda) in the cold-shock expression system versus the T7 system. Figure 3. Expression of Human Gene C. Comparison of expression of soluble human gene C protein (~ 80 kda) in the cold-shock expression system versus the T7 system was performed. The expression level of the target protein in the soluble fraction was dramatically higher in the cold-shock expression system as compared to expression in the T7 system. Time after induction (hours) E.col i proteins other than the desired protein are also labeled T7 pcold Pulse labeling Most of the labeled proteins are the products of the desired gene Figure 4. Pulse Labeling of Human Gene D. A comparative study of pulse labeling of human gene D protein (~12 kda) using the cold-shock expression system and the T7 expression system was performed. In the T7 expression system, proteins other than the target protein were labeled. In the cold-shock expression system, the vast majority of the expressed labeled protein is protein D, indicating specific induction of the target. Recombinant Protein Folding & Expression 177

179 4PROTEIN RESEARCH Recombinant Protein Folding & Expression pcold TF DNA pcold TF DNA Cat.# µg Vector for Protein Expression using the Cold Shock Promoter (cspa) High Yield of Active Protein due to Trigger Factor (TF) Chaperone The pcold TF DNA Vector provides cold shock technology for high yield protein expression combined with Trigger Factor (chaperone) expression to facilitate correct protein folding, thus enabling efficient soluble protein production for otherwise intractable target proteins. pcold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kda) which facilitates co-translational folding of newly expressed polypeptides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pcold TF DNA Vector consists of the cspa promoter plus additional downstream sequences including a 5 untranslated region (5 UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multicloning site (MCS). A lac operator is inserted downstream of the cspa promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are located between TF-Tag and the Multiple Cloning Site (MCS) and function to facilitate tag removal from the expressed fusion protein. E. coli strains can serve as expression hosts. GenBank Accession No. AB Note: Licensing agreement is required. Please see our website at This product is for research purposes only. It must not be used for clinical purpose or in vitro diagnosis. pcold ProS2 DNA Protein Expression Utilizing the Promoter of Cold Shock Gene (cspa) Includes Protein S Fusion Tag to facilitate stability and solubility of target protein Takara s pcold ProS2 DNA is a cold shock expression vector that includes Protein S derived from a Gram-negative myxobacteria Myxococcus xanthus as a solubilization tag. Protein S, (173 aa) is very stable soluble protein present in the spore surface coat of Myxococcus xanthus. Fusing a target gene encoding a protein of interest with the ProS2 Tag, found in pcold ProS2 DNA may induce the stability and solubility of the fusion protein. The pcold ProS2 DNA Vector consists of the cspa promoter plus additional downstream sequences including a 5 untranslated region (5 UTR), a translation enhancing element (TEE), a His-Tag sequence, ProS2 Tag, and a multicloning site (MCS). A lac operator is inserted downstream of the cspa promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are located between ProS2 Tag and the Multiple Cloning Site (MCS) and function to facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. The pcold ProS2 DNA Vector provides cold shock technology for high yield protein expression to facilitate correct protein folding, thus enabling efficient soluble protein production for otherwise intractable target proteins. Purity Contains over 70% double-stranded covalently closed circular DNA (RF I) Confirmed to maintain cloning sites by dideoxysequencing method Shown to cleave at a single site by restriction enzymes Nde I, Sac I, Kpn I, Xho I, BamH I, EcoR I, Hind III, Sal I, Pst I and Xba I pcold TF DNA Vector Map References 1. Qing, G. et al (2004) Nature Biotechnology 22: Gerlined, S., et al (1995) EMBO J. 14: pcold ProS2 DNA Cat.# µg Form: 10 mm Tris-HCl (ph8.0), 1 mm EDTA Chain Length: 5,025 bp Purity: Contains over 70% double-stranded covalently closed circular DNA (RF I). Confirmed by dideoxy sequencing method to maintain cloning sites. Confirmed to be cleaved at a single site by restriction enzymes Nde I, Sac I, Kpn I, Xho I, BamH I, EcoR I, Hind III, Sal I, Pst I and Xba I. : 20 C Amp M13 IG pcold TF DNA (5,769 bp) ColE1 ori cspa 3 UTR Multiple cloning site Factor Xa site Thrombin site HRV 3C Protease site Trigger Factor (TF) His Tag TEE cspa 5 UTR lac operator cspa promoter Notes: Licensing agreement required. This product is for research purposes only. It must not be used for clinical or in vitro diagnosis purposes. lac I 178

180 SPP System (Single Protein Production System) SPP System I Cat.# kit SPP System II Cat.# kit SPP System III Cat.# kit SPP System IV Cat.# kit SPP System I-IV Cat.# kit Convenient for Radioisotope Labeling: up to 90% of newly expressed cellular protein is labeled target protein NMR Analysis Purified Protein Expression Takara Bio, in collaboration with Dr. Masayori Inouye at the University of Medicine and Dentistry of New Jersey, USA, has developed a novel system for generating large amounts of highly pure, soluble protein. The SPP (Single Protein Production) System utilizes a modified version of Takara s cold shock expression vectors along with expression of the endoribonuclease mazf to achieve remarkable protein purity and stable isotopic labeling of target proteins. Using this system, up to 90% of newly expressed protein is labelled recombinant target protein. Principle of the SPP System The mazf protein is a sequence-specific endoribonuclease that specifically cleaves single stranded RNAs at 5 -ACA-3 sequences. Expression of mazf virtually eliminates expression of most cellular mrnas, while allowing synthesis of a target protein (ACA sequences need to be subsituted) for up to 72 hours after induction. The SPP System consists of two co-expressed plasmids: 1) an ACA-less pcold expression vector, for expression of an ACA-less version of the target gene; and 2) a vector carrying the maz F gene, for inducible expression of the mazf protein. This system suppresses host protein expression more effectively than the pcold system alone, and results in higher purity and labeling efficiency for a wider variety of recombinant proteins. This system is markedly well suited for protein structural analysis by NMR (nuclear magnetic resonance) or other technologies. Note: Licensing agreement is required. Please see our website at This product is for research purposes only. It must not be used for clinical purpose or in vitro diagnosis. mrna Interferase -MazF Kit Components Cat.# 3367: SPP System I pcold I (SP-4) DNA 20 µg (0.5 µg/µl) MazF (mrna Interferase ) expression plasmid pmazf DNA 0.5 µg (20 ng/µl) Positive Control pcold I (SP-4) envzb DNA 0.2 µg (20 ng/µl) Cat.# 3368: SPP System II pcold II (SP-4) DNA 20 µg (0.5 µg/µl) MazF (mrna Interferase ) expression plasmid pmazf DNA 0.5 µg (20 ng/µl) Positive Control pcold I (SP-4) envzb DNA 0.2 µg (20 ng/µl) Cat.# 3369: SPP System III pcold III (SP-4) DNA 20 µg (0.5 µg/µl) MazF (mrna Interferase ) expression plasmid pmazf DNA 0.5 µg (20 ng/µl) Positive Control pcold I (SP-4) envzb DNA 0.2 µg (20 ng/µl) Cat.# 3370: SPP System IV pcold IV (SP-4) DNA 20 µg (0.5 µg/µl) MazF (mrna Interferase ) expression plasmid pmazf DNA 0.5 µg (20 ng/ul) Positive Control pcold I (SP-4) envzb DNA 0.2 µg (20 ng/ul) Cat.# 3366: SPP System I-IV Cold Shock Expression Vector for SPP System 20 µg (0.5 µg/µl) pcold I (SP-4) DNA, pcold II (SP-4) DNA, pcold III (SP-4) DNA, pcold IV (SP-4) DNA MazF (mrna Interferase ) expression plasmid pmazf DNA 0.5 µg (20 ng/µl) Positive Control pcold I (SP-4) envzb DNA* 0.2 µg (20 ng/µl) * Expression plasmid prepared by inserting ORF of E. coli-derived protein envzb without ACA sequence into pcold I (SP-4) DNA. Estimated molecular weight of expressed protein: 19.6 kda. References 1. Suzuki, M. et al. (2005) Molecular Cell 18: Zhang, Y. et al. (2004) Journal of Biological Chemistry 280: Zhang, Y. et al. (2003) Molecular Cell 12: Qing, G. et al. (2004) Nature Biotechnology 22: mrna Interferase -MazF Cat. #2415A 1,000 U Application Site-Dependent Cleavage of ssrna MazF is a toxin protein in the toxin-antitoxin module of E. coli. It possesses endoribonuclease activity and specifically cleaves single-stranded RNA at the 5 end of ACA sequences. This enzyme does not cleave double-stranded RNA, double-stranded DNA or single-stranded DNA. This product, mrna Interferase - MazF, is supplied as a fusion protein of E. coli MazF and Trigger Factor (TF) an E. coli chaperone protein. The enzyme is also supplied with a 5X MazF buffer (200mM Sodium phosphate, ph 7.5, 0.05% Tween-20). Source Expressed as a recombinant protein in Escherichia coli Definition of Activity One unit is the amount of enzyme that cleaves 1 pmol of standard substrate (ROX-5 -GATAUACATATCT-eclipse, the underlined region is composed of RNA) for 10 min. at 37 C and ph 7.5. Buffer composition use for activity assay: 20 mm Sodium phosphate, ph 7.5 and 0.05% Tween 20. Form Solution in 20 mm Sodium phosphate (ph 6.0), 0.01% Tween-20, 50% glycerol Purity No other nuclease activities were detected under any of the following conditions as assessed by RNA or DNA electrophoresis patterns. 1. After incubation of 40 pmol of ssrna lacking AC sequences with 20 units of mrna Interferase -MazF for 16 hours at 37 C and ph After incubation of 40 pmol of dsrna with 20 units of mrna Interferase -MazF for 16 hours at 37 C and ph After incubation of 1 mg of ldna- Hind III fragments with 20 units of mrna Interferase -MazF for 16 hours at 37 C and ph 7.5. Note: Licensing agreement required. Please see our website at This product is for research purposes only. It must not be used for clinical purpose or for in vitro diagnosis. References 1. Suzuki, M. et al. (2005) Molecular Cell 18, Zhang, Y. et al. (2004) Journal of Biological Chemistry 280, Zhang, Y. et al. (2003) Molecular Cell 12, PROTEIN RESEARCH 4 Recombinant Protein Folding & Expression 179

181 4PROTEIN RESEARCH Recombinant Protein Folding & Expression Brevibacillus Expression System II Brevibacillus Expression System II HB200 1 kit Brevibacillus choshinensis Electro-Cells HB µl Brevibacillus choshinensis Competent Cells HB µl Expression Vector: pnc-hise DNA HB µg Expression Vector: pnc-hisf DNA HB µg Expression Vector: pnc-hist DNA HB µg Expression Vector: pncmo2 DNA HB µg Expression Vector: pny326-bla DNA HB114 1 µg Expression Vector: pni DNA HB µg Expression Vector: pni-his DNA HB µg Expression Vector: pny 326 DNA HB µg Expression Vector: pncmo2-bla DNA HB µg Features Secretes Proteins Very Efficiently Produces Negligible Amounts of Extracellular Protease so products remain intact in the culture medium Proteins are Produced in Active Forms Easy to Culture and Sterilize Brevibacillus is a Safe Organism Can be Used for Intracellular Production Also Brevibacillus choshinensis is a Gram positive bacterium which has exceptional capacity for heterologous protein expression. The Brevibacillus Expression System II enables highly efficient production of target protein in secreted form. This system allows high yield of active proteins and is well-suited for expression of eukaryotic proteins. The Brevibacillus system is nearly free of proteases, which facilitates production of intact protein products. The Brevibacillus system facilitates disulfide bond formation (commonly required in proteins of eukaryotic orign). In addition, B. choshinensis serves as an excellent host for intracellular protein production, frequently producing intracellul proteins in soluble form in the cytoplasm without forming inclusion bodies. The Brevibacillus system often works better than E. coli for expression of particular targets. Utilizing His-tag containing vectors (pnc-hise, pnc-hisf, pnc-hist, pni-his) allows effective purification of the expressed target protein. Tags can be removed by protease treatment following purification. Kit Components HB200 Brevibacillus Expression System II 1 kit Expression vector pny326 DNA 10 µg pncmo2 DNA 10 µg Control vector pny326-bla DNA 1 µg Competent Cells Brevibacillus choschinesis Competent cells 10 tubes 100 µl MT Medium 10 1 ml Solution A 1 ml Solution B 2 1 ml Notes: License Agreement required. Examples of Proteins Expressed using the Brevibacillus Expression System Proteins Origins Production (g/l) Enzymes α-amylase B. licheniformis 3.7 Sphingomyelinase B. cereus 3.0 Xylanase B. halodurans 0.2 CGTase B. macerans 1.5 Chitosanase B. circulans 1.4 Hyper thermo-stable protease A. pernix 0.1 Hyper thermo-stable nuclease P. horikoshii 0.7 PDI human 1.0 Antigens Surface antigen E. rhusiopathiae 0.9 Surface antigen T. Pallidum 0.8 Cytokines EGF human 1.5 IL-2 human 0.6 NGF mouse 0.2 IFN-γ chicken 0.5 TNF-α bovine 0.4 GM-CSF bovine 0.2 GH flounder

182 Human Cell Free Protein Expression System Human Cell Free Protein System Cat.# X 20 µl reactions pt7-ires His-N DNA Cat.# µg pt7-ires His-C DNA Cat.# µg pt7-ires Myc-N DNA Cat.# µg Application Expression of toxic proteins: that are lethal to host cells of in vivo expression systems Rapid analysis of protein function Rapid analysis of mutation series or truncation series: generate protein quickly and assess functionality using your downstream assay High-throughput proteomic studies Expression of proteins that are easily degraded or insoluble: in conventional in vivo expression systems such as E. coli Features: Easy-to-Use System: allows generation of protein in as little as 1 hr in a singletube reaction Provides Higher Yield of Functional Protein and Greater Consistency: than rabbit reticulocyte or wheat germ in vitro translation systems Excellent with Challenging Proteins: such as large proteins (over 150 kda) and proteins requiring post-translational modification Amenable to High-Throughput Screening: bulk sizes available The Human Cell-Free Protein Expression System from Takara Bio is easy to use. The single-tube reaction is easily assembled and protein synthesis is complete in as little as 1 h at 32 C. The Human Cell-Free Protein Expression System provides high yield (e.g., 50 µg/ml of human eif4g) of functional protein, including proteins requiring modifications such as glycosylation, phosphorylation, or fatty acylation and even with large proteins (over 150 kda). Genes of interest may be cloned rapidly into pt7-ires vectors using In-Fusion cloning technology. After expression, proteins with N-terminal or C-terminal His tag or N-terminal Myc tag can be generated, depending on choice of vector. Bulk sizes are available for high-throughput studies; contact takaracustom@clontech.com for more information. In vitro translation has many advantages for protein expression: it is excellent for rapid studies of protein function or features, amenable to high-throughput studies, useful for proteins that are degraded or insoluble with in vivo systems, and can be used to generate lethal proteins that cannot be expressed using in vivo systems due to toxicity. In vitro translation systems also allow post-translational modifications such as glycosylation, phosphorylation, and fatty acylation. Kit Components 3281 Kit Components (for µl reactions) Cell Lysate*1 100 µl Mixture-1 60 µl Mixture-2*2 10 µl Mixture-3*2 20 µl T7 RNA Polymerase (200 U/µl) 10 µl pt7-ires Vector (0.5 µg/µl) 20 µl Control Vector*3 (0.3 µg/µl) 5 µl *1: Dissolve just prior to use; mix well gently with a micropipette and use immediately. After use, promptly store at -80. Note: Although five cycles of freeze-thaw generally would not lead to any decline in performance, the cell lysate should be stored in aliquots of the required volume. *2: Mixture-2 and Mixture-3 contain protein. To avoid protein deactivation, do not stir excessively or vortex. Mixture-2 contains an HN-tagged protein. *3: This vector harbors a β-galactosidase gene. Bacillus Subtilus Secretory Protein Expression System Bacillus Subtilus Secretory Protein Expression System Cat.# reactions Expression of Soluble, Recombinant Protein: secreted directly into the culture media Protein Expression in a Host Amenable to Medium- and Large-Scale Fermentation: in addition to small-scale culturing Expression of Proteins with Complex Structure: such as proteins with disulfide (S-S) bonds Generation of Target Protein in a Host that is considered to be Generally Regarded As Safe (GRAS) by the U.S. Food and Drug Administration Useful for Producing Easily Purified Recombinant Protein: with proper in-frame cloning, a C-terminal His tag can aid purification from culture media Features: Includes pbe-s DNA, an E. coli/b. subtilis shuttle vector with B. subtilisderived suptiliin (apre) promoter, secretory signal peptide (apre SP), Multi-Cloning Site, and 3 (C-terminal) His-tag sequence Supplied with SP DNA Mixture: a library of DNA sequences encoding 173 unique secretory signal peptides that can be inserted upstream of your target gene Fully Compatible with In-Fusion Cloning Kits: and systems to allow rapid and easy construct generation Includes B. subtilis strain RIK1285 Bacillus subtilis has become an increasingly popular host for recombinant protein expression. With its ability to secrete protein directly into culture media, amenability to medium- and large-scale fermentation, lack of codon bias, and designation by the U.S. Food and Drug Administration as an organism that is Generally Regarded As Safe (GRAS), it s no wonder that the majority of industrially-produced enzymes are expressed in Bacillus species such as B. subtilis. Optimization of secretion, however, can be necessary to achieve highest yields. To address this, the B. subtilis Secretory Protein Expression System from Takara Bio allows rapid development of a library of B. subtilis clones, each bearing a pbe-s construct in which the ORF for your protein of interest is fused with sequences for 173 unique signal peptides. Perform a downstream assay to identify and select clones which secrete the highest amount of functional protein into the culture media, and you can quickly identify the signal peptide that results in efficient expression of your desired secreted protein. Kit Components 3380 SP DNA mixture (0.032 pmol/µl) 45 µl pbe-s DNA (0.5 µg/µl) 20 µg B.subtilis RIK1285 (glycerol stock) µl PROTEIN RESEARCH 4 Recombinant Protein Folding & Expression 181

183 4PROTEIN RESEARCH In vitro Refolding Chaperonin GroE Chaperonin Gro EL Cat.# mg Chaperonin Gro ES Cat.# mg Refolding of Denatured Proteins and Restoration of Activity Chaperonin GroE is a protein complex composed of GroEL (14 subunits, 57 kda) and GroES (7 subunits, 10 kda) and supports proteins in forming their tertiary structure upon (or immediately after) translation. GroE is essential to assembly of protein complexes in vivo. Takara GroE can be used for refolding denatured proteins to recover functional activity. Source Escherichia coli Form Lyophilized equivalent to 5 mg (GroEL) or 0.5 mg (GroES) in 200 µl of 5 mm Tris-HCl buffer, ph 7.8) Quantity GroEL: 5 mg protein/vial GroES: 0.5 mg protein/vial Purity 90% on SDS-PAGE Properties GroEL: Molecular weight (one subunit): 57,000 (amino acid sequence) 60,000 (SDS-PAGE) Isoelectric point: 4.7 Optimum ph: Stable ph range: Optimum temperature: C Thermal stability : stable at < 40 C Tolerance to denaturants: stable in presence of < 100 mm Guanidine-HCl Gro ES: Molecular weight (one subunit): 10,000 (amino acid sequence) 15,000 (SDS-PAGE) Isoelectric point: 5.0 Optimum ph: Stable ph range: Optimum temperature: C Thermal stability: stable at < 40 C Tolerance to denaturants: stable in presence of < 100 mm Guanidine-HCl 20 C 182

184 Protein Disulfide-Isomerase (PDI) Protein Disulfide-Isomerase (PDI) Cat. # mg Refolding of Proteins by Exchange Reactions between Disulfide Bonds Protein Disulfide-Isomerase (PDI, Enzyme Code ) accelerates the exchange reactions between disulfide bonds in proteins in the presence of appropriate oxidizing or reducing reagents. It is especially useful when working with recombinant proteins that sometimes lack the tertiary structure of native proteins are not functional. Source Bovine liver Form Lyophilized white powder (Containing the amount equivalent to 200 μl of 50 mm sodium phosphate buffer, ph 7.5) Volume 1 mg protein/vial 300 U/mg Corystein (Purothionin) Reagent Corystein (purothionin) is a polypeptide purified from wheat endosperm. This prod uct catalyzes the correct formation of disulfide bonds in pro teins. It can be used alone or together with thioredoxin to re-form disulfide bonds on a variety of proteins. Source Wheat flour 20 C Purity Homogeneous on SDS-PAGE Properties Molecular weight: 107,000 (homodimer) Optimum ph: Isoelectric point: 4.2 Definition of Activity One unit of PDI is defined as the amount required to recover one RNase A unit from reduced bovine RNase A, at 25 C in 15 minutes at ph7.5. One RNase A unit is defined as the amount required for hydrolysis of ccmp (cytidine 2 : 3 -cyclic monophosphate) that results in an increase of absorbance unit at 284 nm per minute, at 25 C in one minute, at ph 7.5. References 1. Goldberger, R. F., Epstein, C. J. and Anfinsen, C. B. (1964) J. Biol. Chem., 239, Lambert, N. and Freedman, R. B. (1983) Biochem. J., 213, Hillson, D. A., Lambert, N. and Freedman, R. B. (1984) Methods in Enzymology, 107, Tang, J. G., Wang, C. C. and Tsou, C. L. (1988) Biochem. J., 255, 451. Corystein (Purothionin) Reagent Cat.# mg Protein Refolding through Exchange Reactions between Disulfide Bonds Form Lyophilized powder Purity Homogeneous on SDS-PAGE Properties Molecular weight: kda (calculated) Stable ph range: Isoelectric point: 10.0 Thermal stability: <80 C 4 C PROTEIN RESEARCH 4 In vitro Refolding/C-terminal Isolation & Analysis 183

185 4PROTEIN RESEARCH N-Terminal Deblocking & Analysis Refolding CA Kit Refolding CA Kit Cat.# 7350 (small) 25 reactions Refolding CA Kit Cat.# 7351 (large) 1 kit Application Refolding of Isolated Inclusion Body Proteins The Refolding CA Kit uses a novel artificial chaperone technology (licensed from NFRI, BTRAI, and Ezaki Glico Co, Ltd.) to allow an easy 2-step procedure for determining optimal refolding conditions for inclusion body proteins. Choosing and performing the optimal conditions results in correct protein folding and restoration of protein activity. The small kit is supplied with guanidine hydrochloride and DTT for protein unfolding, four different surfactants that can be added independently to the unfolded protein solution to provide protection against molecular aggregation, and highly polymerized cycloamylose (CA), an artificial chaperone, for surfactant removal and recovery of protein activity. Overnight incubation of the CA-treated protein is followed by a quick 10-minute centrifugation and collection of the supernatant containing the refolded protein. Use large kit for large scale refolding after the reaction conditions have been determined using the small Refolding CA kit. The large kit consists of only denaturant and CA. Related Products Chaperone Plasmid Set, Cat.# 3340 References 1. Machida, S., et al. (2000) FEBS Lett. 486: Sundari, C.S., et al. (1999) FEBS Lett. 443: Daugherty, D.L., et al. (1988) J. Biol. Chem 273: Guanidine hydrochloride unfolds inclusion bodies Surfactants prevent protein aggregation Kit Components 7350 (Small Kit) 8 M guanidine hydrochloride (GdmCl) 2 1 ml 4 M dithiothreitol (DTT) 50 µl 4 surfactants: 1% Tween ml 1% Tween ml 1% CTAB (cetyltrimethylammoniumbromide) 2 1 ml 1% SB3-14 (myristylsulfobetaine) 2 1 ml 200 mm DL-cystine ml 3% CA (highly polymerized cycloamylose) ml 7351 (Large Kit) 8 M guanidine hydrochloride (GdmCl) 2 10 ml 3% CA (highly polymerized cycloamylose) 6 20 ml Inclusion body Suspend in an appropriate buffer. (recommended concentration : 10 mg protein/ml or less) Suspension of inclusion body Add 75 µl of 8 M Guanidine Hydrochloride. Add 1 µl of 4 M DTT. Incubate at room temperature for 1 hour. 20 µl of unfolded protein solution Highly polymerized CA removes surfactants and facilitates protein refolding Add 1.4 ml of surfactant solution* (+DL-Cystine)*. *Containing 70 µl of 1% surfactant (and 14 µl of 200 mm DL-Cystine) and an appropriate buffer. Incubate at room temperature for 1 hour. 400 µl of reaction mixture. Biologically active protein in thermodynamically stable native conformation Add 100 µl of 3% CA. Incubate overnight at room temperature. Centrifuge at 15,000 rpm. Supernatant Refolded protein solution Protocol for Refolding CA Kit Principle of Refolding CA Kit 184

186 Pfu Pyroglutamate Aminopeptidase Pfu Pyroglutamate Aminopeptidase Cat. # mu Removal of Pyroglutamic Acids from the N-termini of Proteins and Peptides Deblocking of N-terminal Pyroglutamates of Proteins and Peptides for Sequence Analysis using Edman Degradation Pfu Pyroglutamate Aminopeptidase liberates the N-terminal pyroglutamic acid from proteins and peptides. This enzyme may work well with some intact, non-denatured protein. In such cases, a denaturation step may be unnecessary. This product is supplied with 5X Reaction Buffer [250 mm sodium phosphate (ph 7.0), 50 mm DTT, 5 mm EDTA]. Systematic name L-Pyrrolidone carboxyl peptidase. Enzyme code: Source Escherichia coli carrying plasmids encoding the Pyrococcus furiosus pyroglutamate aminopeptidase gene. Form Lyophilized Volume 10 mu/vial Purity 90% on SDS-PAGE. No other proteases detected. Pfu Methionine Aminopeptidase Properties Molecular weight: kda (calculated) 28 kda (by SDS-PAGE) Optimum temperature: C Thermal stability: ~90% activity at 75 C + ph 7.0 for 150 min Optimum ph: Stable ph range: % activity in this range (5.0 to 9.0) at 75 C when reacted for 30 min. Tolerance to denaturants: 1 M Urea 1 M Guanidine-HCl 0.01% SDS Inhibitors: PCMB, Hg 2+ Supplied Buffer (5X) Volume: 1 ml Component: 250 mm sodium phosphate buffer (ph 7.0) containing 50 mm DTT and 5 mm EDTA Comparison of specific activities at 37 C, 50 C or 75 C Enzyme with a specific activity of 5.83 U/mg protein at 37 C showed activity equivalent to of 12.2 U/mg protein at 50 C or 28.3 U/mg protein at 75 C, respectively. Definition of Activity One unit of enzyme activity corresponds to the amount required to hydrolyze 1 µmol of pyroglutamate p-nitroanilide at 37 C in 1 minute at ph 7.0. References 1. Shimada, Y. et al. (1989) J. Biochem. 106: Hamazume, Y. et al. (1987) J. Biochem. 101:217. Pfu Methionine Aminopeptidase Cat.# mu Application Release of Met Residue from Recombinant Proteins and Peptides Pfu Methionine Aminopeptidase is a thermostable Methionine Aminopeptidase isolated from Pyrococcus furiosus and produced as a recombinant protein. This enzyme liberates the N-terminal methionine residue from proteins and peptides. Source Escherichia coli carrying plasmids encoding the Pyrococcus furiosus methionine aminopeptidase gene. Form Solution in 10 mm Tris-HCl, ph 7.5, containing 0.01% Tween 20, and 0.1 mm CoCl 2 Volume 20 mu/vial Purity 95% homogeneous on SDS-PAGE. No other proteases are detected. Properties Molecular weight: 32,848 (Mass spectrum analysis) 37,000 (SDS-PAGE) Optimum ph: Stable ph range: This enzyme is stable at ph (75 C, 1 hour, 0.5 mm Co 2+ ) Optimum temperature range: C Thermostability: This enzyme is stable for 1 hour at 75 C (ph 7.2, 0.5 mm Co 2+ ). Tolerance to denaturants: Stable in the presence of 2 M urea 0.2 M guanidine-hcl 0.01% SDS Activator: Co 2+ Inhibitor: EDTA 20 C Note Prior to digestion with this enzyme, it is necessary to denature the protein samples using a chemical procedure such as carboxymethylation. PROTEIN RESEARCH 4 N-Terminal Deblocking & Analysis References 1. Ben -Bassat, A., Bauer, K., et al. (1987) J. Bacteriol., 169, Miller, C. G., Strauch, K. L., et al. (1987) Proc. Natl. Acad. Sci. USA, 84, Ben -Bassat, A., Bauer, K., et al. (1987) Nature, 326, Yasueda, H., Nagase, K. et al. (1990) Bio/Technology, 8,

187 4PROTEIN RESEARCH Pfu Aminopeptidase I Pfu Aminopeptidase I Cat. # mg Application Liberates the N-Terminal Amino Acids up to X-Pro from Proteins and Peptides Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase isolated from Pyrococcus furiosus. It is produced as a recombinant protein, which liberates the N-terminal amino acid from proteins and peptides. This enzyme has a wide range of substrate specificity, and it does not hydrolyze peptide bonds at the a-amino residue side of proline (X-Pro). It is significantly activated in the presence of a Co 2+ ion. Source Escherichia coli carrying plasmids encoding the Pyrococcus furiosus aminopeptidase I gene. Form Lyophilized Purity Homogeneous on SDS-PAGE. Properties Molecular weight: kda (calculated) kda (SDS-PAGE) Isoelectric point: Inhibitor: EDTA (Completely inhibited at 0.1 mm) Optimum ph: (in the presence of 20 µm Co 2+ at 90 C) Optimum Temperature: 80 C (in the presence of 20 µm Co 2+, ph 6.0) 95 C (without Co 2+, ph 6.0) Thermal Stability: The enzyme retains 65% activity after 4 hrs. at 90 C (ph 8.0, without Co 2+ ) Activities Approximately 84 U/mg protein (5 mm Leucine-p-nitroanilide) Approximately 344 U/mg protein (in the presence of 20 µm Co 2+, 5 mm Leucine-p-nitroanilide) Definition of Activity One unit of enzyme activity corresponds to the amount required to hydrolyze 1 µmol of Leucine-p-nitroanilide at 75 C, ph 8.0, in 1 minute. N-Terminal Deblocking & Analysis/Protein Fragmentation Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) Cat.# µg Application Properties Liberates the N-Terminal Acyl Blocking Group from Proteins Molecular weight: 38,639 (mass spectrometry) 451,000 (by sedimentation equilibrium method) Activator: CoCl 2 Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl), and then releases Optimum ph: Inhibitor: Amastatin, EDTA the subsequent amino acids from proteins and peptides until it reaches the first X-Pro Optimum temperature: C Thermal stability: Enzyme retains 100% activity after bond. This enzyme has a wide range of substrate specificities and is used to determine 48 hrs at 50 C in the supplied buffer (ph 8.0 with 0.1 mm Co short stretches of amino acid sequence of blocked proteins and peptides whose ) sequence can be determined by Mass Spectrometry. In addition, since the amino Supplied Buffer (5X) terminus of Ac-DAP is acetylated, its amino acid sequence cannot be determined by Edman degradation. Therefore, even if a high E/S ratio is used (for example, E/S = 0.5 1), the amino acid sequence of the target protein or peptide can still be determined Volume : Component : 1 ml 250 mm N-Ethylmorpholine-AcOH buffer (ph 8.0) containing 0.5 mm CoCl 2 by Edman Degradation without requiring separation from the enzyme. Note: Ac-DAP cannot act upon a non-denatured protein; therefore, the protein sample must be completely denatured by carboxymethylation prior to Ac-DAP digestion. Once thawed, buffer should be stored at 4 C. Avoid freeze-thaw cycles. Specific Activity 8 10 units/mg Source Yeast carrying a plasmid with the gene encoding Pyrococcus furiosus DAP Form Solution in 50 mm N-Ethylmorpholine-AcOH buffer (ph 8.0) Protein Concentration 1 mg/ml Purity Homogeneous on PAGE Definition of Activity One unit of the enzyme activity corresponds to the amount required to hydrolyze 1 µmol of Leucine-p-nitroanilide at 75 C, ph 8.0, in 1 minute. 20 C Once thawed, the enzyme solution should be stored at 4 C. The thawed enzyme and bufffer are stable at least 3 months at 4 C. Note Since this enzyme cannot react with proteins containing higher order structure, protein samples should be denatured by chemical procedures such as carboxymethylation. This enzyme cannot act on proteins immobilized on PVDF membrane

188 Arginylendopeptidase Arginylendopeptidase Cat.# mg Application Fragmentation of Proteins and Peptides Prior to Structural Analysis Arginylendopeptidase cleaves peptide bonds at the carboxyl side of arginine residues neither pro teins and pep tides. Arginylendopeptidase is also known as mouse submaxillary pro tease D or as mouse EGF-binding protein C. Takara s arginylendopeptidase has been treated with TLCK and TPCK to remove trace trypsin-like and chymotrypsin-like protease activities. The product is supplied with 5X Reaction Buffer [250 mm sodium phosphate buffer (ph 8.0)]. Note: The enzyme has weak activity toward -Lys-X- sites, especially when preceded by a basic amino acid residue. Source Mouse submaxillary glands Form Solution in 5 mm sodium phosphate buffer (ph 7.2) con tain ing 50% glycerol Purity Homogeneous on SDS-PAGE. No other proteases detected. Properties Molecular weight: 21.3 kda (gel filtration) Optimum ph: Isoelectric point: 5.65 Inhibitors: PMSF, DFP Tolerance to denaturants: stable in presence of #2 M Urea stable in presence of #0.1 M Guanidine-HCl stable in presence of #0.05% SDS Definition of Activity One unit of enzyme activity corresponds to the amount required to produce 1 mmol of p-nitroaniline from benzoyl-dl-arginine p-nitroanilide (BAPA) in 1 minute at 37 C, ph C References 1. Isackson, P. J. et al. (1987) Biochemistry 26: Matsushita, H. et al. (1988) Frontier Forum on Protein Microsequencing. 3. Matsushita, H. et al. (1989) Protein, Nucleic Acid and Enzyme 34:374. (Japanese Journal) PROTEIN RESEARCH 4 Asparaginylendopeptidase Asparaginylendopeptidase Cat.# mu Application Fragmentation of Proteins and Peptides Prior to Structural Analysis Asparagylendopeptidase specifically cleaves peptide bonds on the carboxyl side of asparagine residues, including Asn-Pro bonds. The substrate specificity of this enzyme has been validated on many peptides and proteins. Peptide bonds next to N-terminal or N-second position asparagine residues will not be cleaved with this enzyme. Bonds adjacent to C-terminal asparagines residues are cleaved. Also, because asparagine residues bound to sugar chains are not cleaved, this enzyme can be used to estimate the attachment sites of sugars in glycoproteins of known sequence. Asparaginylendopeptidase is isolated from Jack bean. Source Jack bean Form Solution in 20 mm sodium acetate buffer (ph 5.0) containing 50% glycerol, 0.005% Brij-35, 1 mm DTT, and 1 mm EDTA Volume 0.2 mu/vial (5 10 uses for digestion of 2 nmol protein) Purity No other proteases detected. Properties Molecular weight: 37,000 (SDS-PAGE) Optimum ph: Stable ph range: Optimum temperature: C Thermal stability: Stable below 50 C Inhibitors: p-chloromercuribenzoate (PCMB) N-ethylmaleimide (NEM) Tolerance to denaturants: stable in presence of 2 M urea stable in presence of 0.5 M guanidine-hcl stable in presence of 0.05% SDS Supplied Buffer (5X) Volume : 1 ml Component : 250 mm Sodium acetate, ph 5.0, 50 mm DTT, 5 mm EDTA Definition of Activity One unit of enzyme activity corresponds to the amount required to produce 1 µmol of DNP-Pro-Glu-Ala-Asn from DNP-Pro-Glu-Ala-Asn-NH 2 in 1 minute at 37 C, ph C Note Prior to digestion with asparaginylendopeptidase, denature the protein samples by chemical procedure such as carboxymethylation. Protein Fragmentation 187

189 4PROTEIN RESEARCH Protein Fragmentation/Protease Inhibition Endoproteinase Asp-N Endoproteinase Asp-N Cat. # µg Application Fragmentation of Proteins and Peptides Prior to Primary Structure Analysis Endoproteinase Asp-N is a metalloprotease that hydrolyzes peptide bonds on the amino side of Asp. It will also cleave at the N-terminal side of Cys residues that have been oxidized to cysteic acid. If cysteine is reduced or alkylated, the enzyme will cleave only the amino side of Asp residues. The enzyme is supplied with 250 mm sodium phosphate buffer (ph 8.0). Source Pseudomonas fragi mutant Form Lyophilized (containing the equivalent of 2µg enzyme in 50 µl of 10 mm Tris-HCl, ph 7.5) Pfu Protease S Pfu Protease S Cat.# U Application Fragmentation of Proteins and Peptides Prior to Primary Structure Analysis Pfu Protease S is an endo-type serine protease with broad activity toward native and denatured proteins. Cleavage occurs primarily on the carboxy side of peptide bonds of hydrophobic amino acid residues. Source Bacillus species carrying a plasmid that encodes the Pyrococcus furiosus protease gene Form: Solution in 25 mm Tris-HCl (ph 7.6) containing 40% ethanol Calpastatin Application Calpain Protease Inhibitor Calpastatin is an endogenous protease in hib i tor that acts specifically on calpain (a calcium-dependent cysteine pro tease). It consists of four repetitive sequences of 120 to 140 amino acid residues (domains I, II, and IV), and an N-ter mi nal non-homologous sequence (L). The prod uct consists of highly purified recombinant human calpastatin domain I. Form: Lyophilized white powder Source: Recombinant Human Calpastatin Domain I Activity Approximately 14 U/µg protein Purity Homogeneous on SDS-PAGE. No other proteases detected. Properties Molecular weight: 27 kda (SDS-PAGE) Optimum temperature: 37 C Optimum ph: Inhibitors: 2-phenanthroline, EDTA, DTT Definition of Activity One unit of enzyme activity corresponds to the amount required to increase absorbance unit of the peptide soluble in trichloro-acetic acid at 280 nm in 1 minute at 37 C, ph 8.0 using casein as the substrate. Purity: Homogeneous on SDS-PAGE. No other proteases detected. Properties Molecular weight: 45 kda (SDS-PAGE) kda (calculated) Optimum temperature: C Optimum ph: Inhibitor: PMSF Tolerance to denaturants at 95 C ph 7.0: 1% SDS; retains 50% activity after 24 hr.s at 95 C, ph M urea; retains 70% activity after 1 hr. at 95 C, ph % acetonitrile; retains 90% activity after 1 hr at 95 C, ph 7.0 Definition of Activity One unit of enzyme activity corresponds to the amount required to hydrolyze 1 mmol of N-succinyl Ala-Ala-Pro-Phe p-nitroanilide in 1 minute at 95 C, ph 7.0. Calpastatin Cat.# mg Purity: Homogeneous on SDS-polyacrylamide gel elec tro phore sis. Properties Molecular weight: Peptide length: 14 kda 137 aa Activity 50 nm of calpastatin domain I completely inhibits the activity of 7.5 µg/ml calpain I. 15 nm of calpastatin domain I inhibits 50% of the activity of 7.5 µg/ml calpain I. : 20 C References 1. Uemori, T. et al. (1990) Biochem. Biophys. Res. Comm. 166: Asada, K. et al. (1989) J. Enz. Inhib. 3: Kanaki, R. and Murachi, T. (1987) Protein, Nucleic Acid and Enzyme 32:116 (Japanese Journal)

190 Glycobiology Glycobiology Related Kits Sialic Acid Fluorescence Labeling Kit Cellulose Cartridge Cloned a2,3-sialidase Lacto-N-Biosidase aα1,2-l-fucosidase aα1,3/4-l-fucosidase aα1,3/4-l-fucosidase Pyridylamination Manual Kit Lysogangliosides regcase II Recombinant (endoglycoceramidase II) EGCase II ACT RE GLYCOBIOLOGY 5 Table of Contents 189

191 5GLYCOBIOLOGY Glycobiology Related Kits Sialic Acid Fluorescence Labeling Kit Sialic Acid Fluorescence Labeling Kit Cat. # assays Application Kit Components Quantitative and Highly Sensitive Sialoglycoconjugates Analysis DMB Solution 2 1 ml Coupling Solution* 2 5 ml *Contains 2-mercaptoethanol, sodium hydrosulfite, acetic acid Sialic acids are important constituents of glycoconjugates in microorganisms and a Neu5Ac (100 µm) 500 µl wide range of higher animals. Many sialic acids are bound to sugar chains through Procedure a-ketoside linkages such as a-2,3, a-2,6 and a-2,8. Sialic acid-rich glycoproteins Set samples containing free sialic acids (ranging from 5 pmol to 5 nmol) into are deeply involved in cell-to-cell recognition, blood coagulation, fertilization and screw-capped 1.5 ml tubes. other biological events. Conventional methods of sialic acid quantification such Note: The sample volume must be kept under 50 µl. Since acidic conditions are as the resorcinol assay and thiobarbituric acid test involve laborious and complex required for the following reaction, the ph must be kept neutral or acidic. procedures. Prepare a mixed solution, with a ratio of Reagent 1:Reagent 2:H 2 O of 1:5:4. Add However, the HPLC-based sialic acid fluorescence labeling technique using 200 µl of this mixed solution to the samples, stir and allow reaction to proceed 1,2-diamino-4,5-methyleneoxybenzene (DMB) is a relatively simple and highly for 2.5 hours at 50 C, while protected from light. sensitive quantitative method. In this method, free sialic acids are analyzed by Note: This mixed solution is stable for up to 1 week at 4 C. reverse phase HPLC (PALPAK Type R) after labeling by DMB. Not only Neu5Ac but Incubate on ice to terminate the reaction. also NeuGc, KDN and their O-acetyl derivatives can be analyzed separately by this method, and the detection limit is <57 fmol. Combining the DMB method with the Inject 10 µl of the reaction mixture into a HPLC column. Quantities of sialic acids pyridylamino (PA) derivatization method allows sialoglycoconjugates to be within the sample can be calculated from the calibration curve based on the successfully analyzed with extremely high sensitivity. peak height obtained with the defined sialic acid standard. Store at 20 C. Store DMB Solution in dark. References 1. Klein, A. et al. (1997) Glycobiology 7: Hara, S. et al. (1989) Anal. Biochem. 179: Nakamura, M. et al. (1987) Chem. Pharm. Bull. 35:687. Experimental Example for the Sialic Acid Fluorescence Labeling Kit DMB Labeling Time Course 200 µl of labeling reagent mixture was added to 10 µl of an aqueous solution containing 10 pmol each of NANA, NGNA and KDN. Incubation: 50 C in a dark place. Sample times: 0.5, 1.25, 2.5 and 5.0 hours. 10 µl of each sample (476 fmol of each type of sialic acid) was analyzed by HPLC. HPLC Conditions Column: PALPAK Type R (4.6 mmf 250 mm) Solvent A: methanol/h 2 O=7/93 (v/v) Solvent B: acetonitrile/methanol=93/7 (v/v) Elution: 0 25 min, 5% Solvent B min, 100% Solvent B min, 5% Solvent B (re-equilibration) Flow rate: 1 ml/min Temperature: 40 C Detection: Fluorescence (ex. 373 nm, em. 448 nm) Isolation of DMB labeled sialic acid. Time course results

192 Cellulose Cartridge Cellulose Cartridge Glycan Preparation Kit Cat.# Kit Cellulose Cartridge Column Pack Cat.# columns Application Clean up of Glycan Samples before Pyridylamination The Cellulose Cartridge Glycan Preparation Kit is designed for clean up of glycan samples before pyridylamination. A schematic diagram of a typical glycan clean-up protocol is shown in Figure A. Cellulose cartridges are prepackaged with carefully prepared fine cellulose microcrystals. Glycans adsorb to the cellulose via hydrophilic interactions during the loading step. Contaminants, such as hydrazinolyzed cell and glyco proteins and enzyme reaction mixtures are removed by washing. Finally, isolated glycans are eluted with ethanol/water. Note: This kit is also effective for removal of residual reagents after pyridylamination. Application Example The figures below show HPLC results from samples with or without clean-up using Cellulose Cartridge Glycan Preparation Kit. Sample a. Hydrazinolyzed Protein No Clean-up Clean-up before Labeling GLYCOBIOLOGY 1. Sample Loading 2. Wash (BuOH/EtOH/W) (BuOH/EtOH/W) 3. Elution (EtOH/W) min 5 Sample b. Hydrazinolyzed Cultured Cells Glycans Contaminants No Clean-up Figure A: Schematic Diagram of a Typical Glycan Clean-up Protocol Kit Components Glycan preparation kit (Cat.# 4403) Cellulose Cartridge Plastic Syringe (10 ml) Adaptor Column Pack (Cat.# 4404) Cellulose Cartridge Properties of Cartridge Adsorbent: cellulose microcrystals Bead volume: 0.5 ml Ambient temperature 5 columns 1 pc. 1 pc. 10 columns Clean-up before Labeling min HPLC Conditions Column: PALPAK Type S (4.6 φ x 250 nm) Solvent A: CH3CN/500 mm AcOH-Triethylamine (ph7.3), 90/10 (v/v) Solvent B: CH3CN/500 mm AcOH-Triethylamine (ph7.3)/h2o, 50/10/40 (v/v/v) Elution: 0-40% Solvent B in 40 min, and % Solvent B in 120 min Flow rate: 1 ml/min Column Temp: 40 C Detection: Fluorescence (Ex.; 310 nm, Em.; 380 nm) Glycobiology Related Kits 191

193 5GLYCOBIOLOGY Glycobiology Related Kits Cloned a2,3-sialidase Cloned a2,3-sialidase Cat.# U Substrate Specificity This sialidase has a 260-fold kinetic preference for the a2,3 sialyl linkage. The enzyme efficiently cleaves sialyl groups from gly co pro teins and glycolipids. The enzyme may not release sialic acid from O-linked oligosaccharides on glycoproteins. Systematic Name Acylneuraminyl hydrolase (E.C ) Source E. coli (cloned from Salmonella typhimurium LT2) Form Lyophilized powder (contains 5 µmol of potassium phos phate, ph 6.8, 10 µmol of sodium chloride, and 0.02 mg of sodium azide) Lacto-N-Biosidase Substrate Specificity This enzyme specifically hydrolyzes oli gosac cha rides with type I chains and produces lacto-n-biose (Galb-3GlcNAc). The enzyme does not hydrolyze oligosaccharides with type II chain. The structure of the substrate essential for the en zyme ac tiv i ty is the terminal lacto-n-biosyl residue (Galb1-3GlcNAc). The enzyme does not hydrolyze a2,3-sialyllacto-n-tetraose, lacto-n-fucopentaose I and II. Source Streptomyces sp. 142 Concentration 1 μu/μl Form Solution in 50 mm sodium acetate buffer, ph 5.5, con tain ing 0.05% Brij 58 Properties Molecular weight: K m (substrate): Inhibitors: Optimum ph: 41.3 kda (SDS-PAGE) 0.25 mm (4MUNeuAc) 1.52 mm (3 -sialyllactose) 2.66 mm (PA-3 -sialyllactose) 2-deoxy-2,3-didehydro-Nacetylneuraminic acid K i = 0.38 mm ph (Tris-maleate buff er) ph (potassium phosphate buff er, sodium acetate buff er, Tris-HCl buffer) Definition of Activity One unit is the amount of enzyme required to hydrolyze 1 µmol of 3 - sialyllactose in 1 minute at 37 C, ph 5.5. Lyophilized powder is stable for 12 months at 20 C. Reconstituted enzyme solution is stable for at least 6 months at 4 C. Avoid freeze-thaws. References 1. Hoyer, L.L. (1992) Mol. Microbiol. 6: Hoyer, L.L. (1991) J. Biochem. 110: Lacto-N-Biosidase Cat.# µu Properties Molecular weight: K m (substrate): Optimum ph: Range of ph stability: 60 kda (SDS-PAGE) 6.80 µm Pyridylamino (PA-lacto-N-tetraose) 38.9 µm (PA-oligosaccharide c) ph 5.5 (sodium acetate buffer) ph (4 C, 16 hrs.) Definition of Activity One unit is the amount of enzyme required to hydrolyze 1 µmol of PA-lacto-Ntetraose in 1 minute at 37 C, ph 5.5. The enzyme solution of 1 mu/ml is stable for at least 12 months at below 4 C. Reference 1. Sano, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:

194 α1,2-l-fucosidase α1,2-l-fucosidase Cat.# U Substrate Specificity Properties Specifically cleaves the a1,2 fucoside bond. Liberates L-fu cose from 2 2 -a-lfucosyl lactose (Fuc a1-2 Gal b1-4 Glc) and from oligosaccharides from porcine gastric mucin. Molecular weight: K m (substrate): Inhibitors: 43 kda (gel filtration) M (p-np-fucose) Ag +, Hg 2+, Cu 2+ Optimum ph: ph 8.5 Systematic Name (37 C, 10 min.) a-l-fucoside fucohydrolase (E.C ) Optimum temperature: 34 C Source (ph 8.5, 10 min.) Corynebacterium sp. Range of ph stability: ph (25 C, 60 min.) Thermal stability: #26 C (ph 8.0, 60 min.) Form Lyophilized (white non-crystalized powder) product containing potassium phosphate, EDTA, and sodium azide α1,3/4-l-fucosidase α1,3/4-l-fucosidase Cat.# µu Substrate Specificity Specifically cleaves a1,3 and a1,4 fucoside bonds. Lib er ates L fucose from lacto-n-fucopentaose II, lacto-n-fucopentaose III and N-acetyllactosamine type tri-antennary and tetra-antennary sugar chain with a1,3 fucoside bonds. Also lib er ates L-fucose from a1-acid glycoprotein. Systematic Name a-l-fucoside fucohydrolase (E.C ) Source Streptomyces sp. 142 Form Solution in 50 mm potassium phosphate buffer (ph 6.0) con tain ing 0.1 M NaCI, 0.02% NaN 3, and 0.1% Brij-58 Concentration 1 mu/ml Definition of Activity One unit is the amount of enzyme required to hydrolyze 1 µmol of p-nitrophenyl a-l-fucoside (p-np-fucose) within 1 minute at 37 C at ph 8.5. Store at a temperature less than 4 C in the presence of dessicant. A stock solution of 20 U/mL is stable for at least 3 months at 4 C. The stock solution may be frozen and thawed several times without loss of activity. References 1. Sano, M. et al. (1992) J. Biol. Chem. 267: Sano, M. et al. (1991) Glycoconjugate J. 8:271. Properties Molecular weight: 40 kda K m (substrate): Substrate 1: 10.1 µm (PA-lacto-N-fucopentaose II) Substrate 2: 1.08 µm (PA-lacto-N-fucopentaose III) Optimum ph: ph 6.0 (Substrate 2) Range of ph stability: ph (37 C, 60 min.) Stabilizer: 0.1% NP-40, 0.1% Brij-58, and 0.02% BSA Definition of Activity One unit is the amount of enzyme required to hydrolyze 1 µmol of pyridylamino lacto-n-fucopentaose III within 1 minute at 37 C at ph 6.0. Store at 20 C. The stock solution is stable for at least 12 months at 4 C. Avoid repeated freeze-thaw cycles. Reference 1. Sano, M. et al. (1992) J. Biol. Chem. 267: GLYCOBIOLOGY 5 Glycobiology Related Kits 193

195 5GLYCOBIOLOGY Pyridylamination Manual Kit Pyridylamination Manual Kit Cat.# kit (20 reactions) Reagents required but not supplied in the kit: Fluorescence Labeling of Sugar Chains Containing 2-aminopyridine Butanol (Purity: high grade) approx. 500 ml Ethanol (Purity: high grade) approx. 500 ml Acetic acid (Purity: high grade) approx. 3 ml Ammonium bicarbonate (75 mm aqueous solution) approx. 500 ml This kit is designed to enable fluorescence labeling of sugar chains containing Sterilized distilled water 2-aminopyridine without using special instrumentation. The reduced termini residue of sugar chain 2-aminopyridine is bound in a reduced amino reaction. Package 1: 20 C Kit Component Package 2: room temperature Package 1 References 2-Aminopyridine mg Borane-dimethylamine complex mg 1. Nakamura, M., Hara, S., Yamaguchi, Y., Takemori, Y. and Ohkura, Y. (1987) Chem. Pharm. Acetic acid 3 ml Bull., 35, Hara, S., Yamaguchi, M., Takemori, Y., Furuhata, K., Ogura, H. and Nakamura, M. (1989) Biantennary sugar chain 500 pmol/50 μl Anal. Biochem., 179, 162. PA-Biantennary sugar chain 100 pmol/100 μl 3. Klein, A., Diaz, S., Ferreira, I., Lamblin, G., Roussel, P. and Manzi, A. E. (1997) Glycobiology, Package 2 7, No. 3, Cellulose Cartridge Column ml Adaptor 1 piece Limited Use Label License No. : [M14] Syringe 3 10 ml Glycobiology Related Kits Lysogangliosides Lyso-GM1 Cat.# mg Lyso-GM2 Cat.# ,1mg Lyso-GM3 Cat.# ,1mg Lyso-GD1a Cat.# ,1mg Structure Lyso-GM1 Gal β1-3galnac β1-4gal β 1-4Glc β 1-1'sphingosine 3 Neu5Ac α 2 Lyso-GM2 GalNAc β 1-4Gal β 1-4Glc β1-1'sphingosine 3 Neu5Ac α 2 Lyso-GM3 Gal β 1-4Glc β 1-1'sphingosine 3 Neu5Ac α 2 Lyso-GD1a Gal β1-3galnac β1-4gal β1-4glc β1-1'sphingosine 3 3 Neu5Ac α 2 Neu5Ac α 2 Lyso-gangliosides are prepared by enzymatically removing glycolipid from natural gangliosides. They can be used to analyze various cell functions, such as cell proliferation, differentiation, apoptosis, signal transduction, etc. They can also be used as a sample of ganglioside derivatives in RI labeling, fluorescence labeling, immobilization since they have free amino group in sphingosine. Purity > 95% by TLC (Solvent; chloroform : methanol : 10% acetic acid=5 : 6 : 2 (v/v/v), Spray; orcinol/sulfuric aicd, sulfuric acid/dichromate) Form Lyophilized 20 C Preparation Each of these products was prepared from bovine ganglioside GM1, GM2, GM3 and GD1a by the action of sphingolipid ceramide N-deacylase (SCDase) (Cat.# 4462). Lyso-GM1 Bovine brain ganglioside GM1 Lyso-GM2 Human brain ganglioside GM2 Lyso-GM3 Bovine milk ganglioside GM3 Lyso-GD1a Bovine brain ganglioside GD1a Properties The structure of sugar chain and sphingosine are the same as those of GM1, GM2, GM3 and GD1a originated from bovine brain respectively. Related Product SCDase (Cat.# 4462) 250mU 194

196 SCDase (Sphingolipid ceramide N-deacylase) SCDase (Sphingolipid ceramide N-deacylase) Cat.# mu Properties For Hydrolysis of N-acyl Linkage Between Fatty Acids and Sphingosine Inhibitors: Hg 2+, Zn 2+ and Cu 2+ Bases Not inhibited by: Mn 2+, Ca 2+, Mg 2+ or EDTA Optimum ph: Hydrolysis ph (0.8% Triton X-100) Reverse reaction ph 7.0 (0.1% Triton X-100) SCDase (Sphingolipid ceramide N-deacylase) from Pseudomonas species hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids. The enzyme also catalyzes the reverse reaction and possesses transacylation activity. The enzyme acts on various acidic and neutral glycosphingolipids and sphingomyelin; however, it exhibits low activity on ceramide. Source Pseudomonas sp. Form Solution in 50 mm sodium acetate (ph 6.0) containing 0.1% Lubrol PX Definition of Activity One unit is the amount of enzyme required to catalyze the hydrolysis of 1 µmol of asialo GM1 per minute. Store at 20 C until use. Store reconstituted solution in aliquots at 20 C. Avoid freeze-thaw cycles. References 1. Mitsutake, S. et al. (1998) J. Biochem. (Japan) 123: Tani, M. et al. (1998) Anal. Biochem. 263: Mitsutake, S. et al. (1997) Anal. Biochem. 247: Sueyochi, N. et al. (1997) J. Lipid Res. 38: Ito, M. et al. (1995) J. Biol. Chem. 270: GLYCOBIOLOGY 5 regcase II Recombinant (endoglycoceramidase II) regcase Recombinant (endoglycoceramidase II) Cat.# mu Structural Studies of Glycosphingolipids Endoglycoceramidase II (EGCase II) cleaves the linkage between the oligosaccharide and ceramide of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. This enzyme does not act on galactosyl- and glucosyl-ceramides, glycoglycerolipids, or glycoproteins. Note: This product contains detergents and is not active without them. Do not use on living cells. Systematic Name Oligoglycosylglucosylceramide glycohydrolase (EC ) Source Recombinant E. coli encoding the gene EGCase II produced by Rhodococcus sp. Concentration 100 mu/50 µl Form Solution in 20 mm sodium acetate (ph 6.0), 0.2% BSA, 0.1% Lubrol PX Purity regcase is free of the following exoglycosidase activities: a-galactosidase, b-galactosidase, a-n-acetylgalactosaminidase, b-n- acetylgalactosaminidase, b-n-acetylglucosaminidase, a-mannosidase, a-fucosidase, and sialidase. This enzyme is also free from glycopeptidase, endo-b- N-acetylglucosaminidase, proteinase, and sphingomyelinase activities. Properties Optimum ph: Inhibitors: 1 mm Hg 2+, Zn 2+, and Cu 2+ Not inhibited by: Ba 2+, Ca 2+, Mg 2+, or EDTA Definition of Activity One unit of enzyme activity is defined as the amount needed to catalyze the hydrolysis of 1 µmol asialo GM1 per minute. Store at 20 C until use. Thawed solution is stable for 2 3 days at 4 C. Glycobiology Related Kits 195

197 EGCase II ACT RE EGCase II ACT RE Cat.# mu 5GLYCOBIOLOGY Notes Cleaves the Linkage between the Oligosaccharide and Ceramide of various Acidic and Neutral Glycosphingolipids Degrades Cell Surface Glycosphingolipids of Living Cells Systematic name of enzyme Oligoglycosylglucosyl-ceramide glycohydrolase Enzyme code EC Source Expressed in Rhodococcus elythropolis carrying a plasmid encoding the EGCase II gene of Rhodococcus sp. Concentration 100 mu/100 µl (containing 50 nmol Activator II) Purity This enzyme is confirmed to be free from the following exoglycosidase activities: α-galactosidase β-galactosidase α-n-acetylgalactosaminidase β-n-acetylgalactosaminidase β-n-acetylglucosaminidase α-mannosidase α-fucosidase sialidase It is also confirmed to be free from endo-β-n-acetylglucosaminidase, glycopeptidase, proteinase and sphingomyelinase activities. Substrate specificity Cleaves the linkage between oligosaccharides and ceramides of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. 1,2 By using EGCase II with the assistance of its protein activator, activator II, cell surface glycosphingolipids of living cells are hydrolyzed without damaging other cell components. 3-6 EGCase II ACT RE is mixture of highly purified recombinant EGCase II and activator II. EGCase II ACT RE is powerful tool for degrading the cell surface glycosphingolipids and is useful for analysis the biological function of cell surface glycosphingolipids. 7-9 EGCase II ACT RE does not hydrolyze cerebrosides, sulfatides, sphingomyelin, glycoglycerolipids and glycoprotein. The galactosylceramide linkage of galatype glycosphingolipids are not hydrolyzed. (Table 1) Cell-surface globo-type glycosphingolipids are strongly resistant to hydrolysis by EGCase II ACT RE. Application example Living cells ( cells) were cultured in 200 µl of modified Eagle's medium (MEM) containing 20 mu EGCase II ACT RE (20 µl) and 5% FCS at 37 C for 6 16 hrs. in a CO 2 incubator. When increasing the reaction volume, also increase the volume of EGCase II ACT RE according to the culture volume. Store at or below 20 C until use. Repeated freeze-thaw cycles results in a loss of enzyme activity. Keep the solubilized enzyme at 4 C and use the recombinant endoglyco ceramidase II within 2 3 days. Definition of activity One unit (U) of enzyme is defined as the amount needed to catalyze the hydrolysis of µmol of asialo GM1 per minute. Properties Optimum ph range: ph 5.5 (32% of activity remaining at ph7-7.5) Inhibitors: The enzyme is inhibited by 1 mm of Hg 2+, Zn 2+ and Cu 2+, but not by Ba 2+, Ca 2+, Mg 2+ and EDTA. 10% (v/v) fetus calf serum (FCS) decreases activity by 50%, while 5% FCS have no effect

198 Instrumentation Thermal Cycler Dice TaKaRa PCR Thermal Cycler Dice TaKaRa PCR Thermal Cycler Dice Mini Mupid Electrophoresis Instruments Mupid One Mupid Related Products INSTRUMENTATION 6 Table of Contents 197

199 6INSTRUMENTATION Thermal Cycler Dice TaKaRa PCR Thermal Cycler Dice TaKaRa PCR Thermal Cycler Dice (Gradient Model) Cat.# TP600 1 unit TaKaRa PCR Thermal Cycler Dice (Standard Model) Cat.# TP650 1 unit Features Sample Setting is Easy Through use of a Slide and Lever Type Cover Handle Easy Viewing and Access in Any Bench: The control panel can be set at a desirable angle, providing: Easy Programming due to excellent graphical user interface Simple Operation using an easy to understand menu and alphanumeric key input method, data entry is similar to a cell phone Easy Implementation of PCR reactions due to eight preinstalled protocols (PCR programs) No Heat Interference with Other Equipment Extension Time Function suitable for long PCR Extension Temperature Function allows touch-down PCR Gradient Function (only for TP600) enables examination of different annealing temperatures The TaKaRa PCR Thermal Cycler Dice is a GRADIENT thermal cycler (only for TP600) which allows temperature variation up to a 20 C slope between the right and left sides of the sample block in each temperature setting step. This allows optimization of reaction conditions to be performed in a single experiment. In addition, the use of a variable angle wing panel combines the control panel and display, making the Dice compact and functional. It requires little space and can be placed anywhere in the lab. Specifications Dimensions Weight Power requirement/consumption Heating cooling system Ramp rate of Heating Ramp rate of Cooling Block temperature control range Block temperature accuracy Block temperature uniformity Exaggerated shot 260 (W) 345 (D) 260 (H) mm (with panel closed) 11.5 kg (System Weight) V, 50/60Hz of AC/ 490 W peltier element Maximum 3.0 C/sec. Maximum 2.0 C/ sec C ±0.5 C ±0.5 C Heated lid 110 C Temperature extension 1 Time extension 1 Sample capacity Display Max. programs stored Max. user name registrations GRADIENT function 2 Less than 0.5 C maximum of ±9.9 C maximum of ±9.59 minutes ml Tube or 96 well PCR Plate Blue/white dot matrix LCD \ ( dots) 100 programs 10 users Range which can be set up: C (temperature variance : 6 20 C) 1 Touch down PCR 2 GRADIENT is set with the first and 12th rows (right end and left end rows) of heat block (96 wells). GRADIENT function is available only for TP600. Application Example Gradient PCR Using the TaKaRa PCR Thermal Cycler Dice (TP600) System Amplification of 8-kb fragment was performed using either in shuttle PCR or conventional PCR using λ DNA template Reaction Mixture TaKaRa Ex Taq (5 units/µl) 0.25 µl 10X Ex Taq Buffer 5 µl dntp Mixture (2.5 mm each) 4 µl Primer 1 (20 µm) 0.5 µl Primer 2 (20 µm) 0.5 µl λ DNA 1 µl Sterilized water µl Total volume 50 µl PCR Conditions Shuttle PCR 98 C 10 sec. 52 to 72 C 5 min. (Gradient temperature : 20 C) 30 cycles Conventional PCR 98 C 10 sec. 45 to 65 C 30 sec. (Gradient temperature: 20 C) 5 min. 72 C 5 min. 30 cycles 198

200 Results: Gradient PCR with TaKaRa PCR Thermal Cycler Dice (Gradient Model) Results for Shuttle PCR 2 step PCR annealing/extension temperature 52 C 72 C M (gradient) M Results for Conventional PCR 3 step PCR annealing temperature 45 C 65 C M (gradient) M INSTRUMENTATION M : λ Hind III marker M : λ Hind III marker Higher annealing/extension temperatures result in more specific amplification and increased yield of target amplicon. 6 Experiment 2: One-Step PCR with TaKaRa PCR Dice and TaKaRa PCR Thermal Cycler SP One Step RT-PCR The performance of the TaKaRa PCR Thermal Cycler Dice (Cat.# TP600) was evaluated in One Step RNA PCR by using the TaKaRa One Step RNA PCR Kit (Cat.# RR024A) and the TaKaRa PCR Thermal Cycler SP (Cat.# TP400). RT-PCR was performed both with and without AMV RTase XL in the reaction mixture. Reaction Mixture 10X One Step RNA PCR Buffer 5 µl 25 mm MgCl 2 10 µl dntp Mixture (10 mm each) 5 µl RNase inhibitor (40 U/µL) 1 µl AMV RTase XL (5 U/µL) or DEPC treated water 1 µl AMV Optimized Taq (5 U/µL) 1 µl Control F-1 Primer 1 (20 µm) 1 µl Control R-1 Primer 2 (20 µm) 1 µl Positive Control RNA 1 µl RNase Free H 2 O 24 µl Total Volume 50 µl PCR Conditions 50 C 15 min. (RT Reaction) 94 C 2 min. (inactivation of RTase) 94 C 30 sec. 60 C 30 sec. 30 cycles 72 C 1.5 min. Results Lane Sample 1 RTase (-) 2 RTase (+) 3 RTase (-) 4 RTase (+) M 100 bp DNA Ladder Dice (TP600) SP (TP400) M 1 2 M 3 4 After PCR, a portion of reaction was visualized using agarose gel electrophoresis. In both cases, using TaKaRa PCR Thermal Cycler Dice (Cat.# TP600) and TaKaRa PCR Thermal Cycler SP (Cat.# TP400), the control reaction 462 bp product was detected only in the presence of RTase (+). Thermal Cycler Dice 199

201 TaKaRa PCR Thermal Cycler Dice Mini Thermal Cycler Dice Mini Cat.# TP100 1 unit 6INSTRUMENTATION The affordable, reliable Takara PCR Thermal Cycler Dice mini is a 24-well block system perfect for any laboratory. Features The Program Link Function: runs programs in numerical order if the program names are the same and # number is entered at the end of the names, enables a complex array of conditions to be set Up to 22 User Names can be Registered: and up to 200 programs can be stored Programming is Easy: due to a graphical user interface Operation is Simple: by utilizing the pop-up menu and alphanumeric key input method, similar to a cell phone PCR Reaction can be Started Easily: due to 8 preinstalled PCR protocols No Heat Interference Between Pieces of Equipment Extension Time Function: suitable for long PCR Extension Temperature Function: allows touch-down PCR Space-saving Design for Personal Use: the Dice Mini is narrower in width compared to conventional systems, saving space on the benchtop (TP600/ TP650: 260 mm in width ; TP100: 210 mm in width) Specifications Product Name Model No. Temperature accuracy Temperature uniformity Temperature setting range Time setting range Heating cooling system Ramp rate of heating Ramp rate of cooling Tubes and plates PCR reaction volume settings TaKaRa PCR Thermal Cycler Dice Mini TP100 +/- 0.5 C (30.0 C to 99.9 C) +/- 2.0 C (4.0 C to 15.0 C) +/- 0.5 C (30.0 C to 99.9 C) 4.0 C to 99.9 C 1 second to 99 minutes, 59 seconds, (up to 9999) Peltier element Maximum 3.0 C/sec. Maximum 2.0 C/sec. 0.2 ml 24 tubes 8 Strip of Tubes (0.2 ml) 3 5 µl to 150 µl Thermal Cycler Dice Program capacity Lid temperature Added functions User names: up to 22 (excluding Common and TaKaRa ) Number of programs: max. 200 Approx. 110 C for the 20.0 C to 99.9 C range Approx. 40 C for the 4.0 C to 19.9 C range Slope function, Program link History function Diagnosis function PC communications (RS-232) (PC transmission software and R5232c cable are not provided) Display Blue/white dot matrix LCD ( dots) Power switch with 5 A over-current breaker Safety devices Lid Heater temperature fuse (169 C) Thermal guard for reaction block heater plate (85 C) Power source 100 to 240V AC, 50/60 Hz, 180VA Dimensions 210 (W) 370 (D) 260 (H) mm Weight 7.5 kg 200

202 Mupid One Mupid -One Cat.# AD160 1 set The Mupid -One electrophoresis system is CE marked. It has novel features including separated power and drain, dimensions that accommodate multichannel pipettes, 7 output voltage settings (18, 25, 35, 50, 70, 100, and 135V) and timer function. This product is manufactured by ADVANCE. Mupid is a registered trademark of ADVANCE. Features Compatible with Multichannel Pipettes. The Mupid -One allows simultaneous electrophoresis of 104 samples on a single gel (26 wells 4 rows (96 samples + 8 markers)). The Mupid -One is Wider than the Conventional Mupid Series and can support 13 or 26 lanes, enabling sample loading with multipipettors (9 mm pitch for 13 wells, 4.5 mm pitch for 26 wells). Voltage Range: Supports seven conventional voltages (18V, 25V, 35V, 50V, 70V, 100V and 135V). Global Input Voltage Compatibility: variable input voltage of 100 to 240V supports use anywhere in the world Heat-Resistant Materials: A liquid gel solution up to 100 C can be poured into the Gel Tray. Clean up can be performed using boiling water. Lengthy Electrophoresis Possible: wide range for voltage control to improve buffer stability during the run. Components One gel casting stand-hr (1)* Several comb setting grooves are provided in the stand. Supports a maximum of four combs per gel with easy position changed. One-Gel Tray L-HR (1) Accommodates up to four rows of wells (running length of 2.7 cm), running up to 104 samples (26 4) is possible. One-Gel Tray S-HR (2) and center partition (1) Used for making 13 and 26 sample gels. One-Comb-HR (4) Four combs can be used for L tray, whereas one comb can be used for S tray. * Number in brackets indicate number of pieces supplied with the chambers Specifications Electrophoresis Cell Overall dimensions 183 mm (W) 59 mm (H) 162 mm (L) Material PPHOX Solution volume Approx ml (includes gel tray and gel) Multi-sample Multi-channel pippette compatible Safety Lid Overall dimensions 197 mm (W) 38 mm (H) 1709 mm (L) Safety Interlock system Without the lid, main power cannot be operated. Power Supply Overall dimensions 75 mm (W) 62 mm (H) 170 mm (L) Weight 410 g Input Voltage AC V, (Internationally compatible) 50/60Hz Output Voltage 135V, 100V, 50V, 25V, 70V, 35V, 18V Constant peak voltage of 140V, and duty-control Timer Timer operation 0 99 min, and continuous mode Temporary shutdown supported Memory Function Automatic memory (the last used V and T) One-Gel Tray Gel tray (small) 130 mm (W) 16.5 mm (H) 59.5 mm (L) Gel tray (large) 130 mm (W) 24 mm (H) 122 mm (L) Quantity small: 2, large: 1 Material Heat Resistant Material (around 100 C of agarose available) One Comb HR Multiple number of wells Wells (13 wells: 9 mm spacing; 26 wells: 4.5 mm spacing) Quantity 4 combs (support making 13 or 26 wells) Material Heat Resistant Material (around 100 C of agarose available) One Gel Casting Stand -HR (makes 2 small gel trays or 1 large gel tray) Overall dimension 140 mm (W) 20 mm (H) 125 mm (L) INSTRUMENTATION 6 Mupid Electrophoresis Instruments 201