Tools for PCR AMPLIFY PURIFY ANALYZE

Transcription

1 Tools for PCR AMPLIFY PURIFY ANALYZE

2 PCR reagents from Affymetrix Affymetrix offers a wide selection of USB PCR tools for robust, accurate, PCR applications to amplify and purify DNA. AMPLIFY We offer the reliable products you need for end point, real-time, hot start, long and accurate PCR, multiplex, qpcr, as well as direct PCR for difficult sample types. USB products for PCR provide the consistent performance you d expect to achieve quality results. PURIFY The ExoSAP-IT family of products are the single-step enzymatic solution for PCR purification without sample loss. Save time and money by eliminating spin columns. ANALYZE Our large selection of enzymes provides you with tools you need for the analysis of downstream applications. Many of the buffers and stock solutions used to analyze PCR products are available in a ready-to-use form. At Affymetrix, we have everything you need for PCR. We test and re-test our reagents to ensure that they work reliably the first time, every time. We can also provide custom or bulk enzymes, formulated to your specific requirements. Visit us at our website at for additional product information, technical references, and protocols. Table of contents Amplify PCR product selection guide Taq polymerase product selection guide End point PCR End point direct PCR End point hot start PCR End point RT-PCR Ultapure nucleotides Multiplex PCR Real-time qpcr Real-time reverse transcription PCR (qrt-pcr) Purify PCR product cleanup PCR purification Nucleic acid purification Analyze Next Generation sequencing products Mutagenesis Sequenase enzyme and kits Thermostable sequencing enzymes and kits Modifying enzymes PCR related reagents OEM, custom, and bulk reagent solutions

3 AMPLIFY PCR product selection guide Let our simple chart below help you find the optimal reagent for your research needs. Navigate our PCR product portfolio by application to view your options. Taq DNA Polymerase [71160] Taq PCR Master Mix (2X) [71162] VersaTaq Direct PCR Polymerase (Inhibition Resistant) [71002] RubyTaq DNA Polymerase [71190] RubyTaq PCR Master Mix (2X) [71191] FideliTaq DNA Polymerase [71180] FideliTaq PCR Master Mix (2X) [71182] VeriQuest Taq DNA Polymerase [71170] HotStart-IT Taq DNA Polymerase [71195] HotStart-IT Taq Master Mix (2X) [71196] HotStart-IT FideliTaq DNA Polymerase [71155] HotStart-IT FideliTaq PCR Master Mix (2X) [71156] MagniTaq Multiplex PCR Master Mix [71199] End point PCR Difficult sample types Direct PCR in blood/tissue/soil Hot start PCR Long and accurate PCR Multiplex PCR Fast PCR Tracking dyes PCR Master mix format Stand-alone enzyme RT-PCR Real-time PCR: SYBR Real-time PCR: Probe One-Step qrt-pcr: SYBR One-Step qrt-pcr: Probe VeriScript Reverse Transcriptase [78070] M-MLV Reverse Transcriptase [78306] AMV Reverse Transcriptase [70041] RT-PCR Master Mix (2X) [78370] FideliTaq RT-PCR Master Mix (2X) [71185] Tth DNA Polymerase [70052] First-Strand cdna Synthesis Kit for Real-Time PCR [75780] VeriQuest SYBR Green qpcr Master Mix (2X) [75600] VeriQuest SYBR Green qpcr Master Mix with Fluorescein (2X) [75665] VeriQuest Probe qpcr Master Mix (2X) [75650] VeriQuest Probe qpcr Master Mix, No Reference Dye (2X) [75660] VeriQuest Fast SYBR Green qpcr Master Mix (2X) [75690] VeriQuest Fast SYBR Green qpcr Master Mix with Fluorescein (2X) [75675] VeriQuest Fast Probe qpcr Master Mix (2X) [75680] VeriQuest Fast Probe qpcr Master Mix, No Reference Dye (2X) [75685] VeriQuest SYBR Green One-Step qrt-pcr Master Mix (2X) [75705] VeriQuest SYBR Green One-Step qrt-pcr Master Mix with Fluorescein (2X) [75715] VeriQuest Probe One-Step qrt-pcr Master Mix (2X) [75700] VeriQuest Probe One-Step qrt-pcr Master Mix, No Reference Dye (2X) [75710] 3

4 AMPLIFY Taq polymerase product selection guide Review our chart below to help you find the optimal Taq polymerase for your research needs. Product name Product no. Description Properties Exonuclease activity Proof-reading activity Taq DNA Polymerase Thermostable Taq DNA polymerase for routine PCR up to 6 kb. Comes with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 5 units/µl 3. Functionally tested in standard PCR 5' 3' No VersaTaq Direct PCR Polymerase Mutant Taq DNA polymerase resistant to common PCR inhibitors, allowing for direct PCR amplification, thus eliminating the need for costly extraction and purification steps. Provided with 10X PCR reaction buffer. 1. Source: Recombinant (E. coli) 2. Concentration: 1.0 ± 0.2 μg/μl, determined by OD Resistance to potent PCR inhibitors in a wide range of end point PCR applications: Whole blood (up to 40%) Feces Blood spots on sample Urine collection cards Animal tissue Buccal swabs Plant tissue Soil Crude extracts 5 3 No RubyTaq DNA Polymerase Taq DNA polymerase containing tracking dye for easy-to-see, easy-to-load convenience. Provided with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 1 unit/µl 3. Inert dyes: A magenta dye runs ~500 bp (on 2% gels) or ~1500 bp (on 0.8% gels), and a yellow dye runs smaller than 10 bp. 4. Functionally tested in standard PCR 5' 3' No FideliTaq DNA Polymerase Combination of Taq DNA polymerase and a high-fidelity, proofreading polymerase for long and accurate PCR. Provided with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 5 units/µl 3. Fidelity 6X greater than standard Taq 4. Generates 3:1 A-tailed to blunt ends 5. Functionally tested to PCR amplify a 20.7 kb product Yes HotStart-IT FideliTaq DNA Polymerase Hot start FideliTaq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific, long and accurate PCR. Provided with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 2.5 units/µl 3. No additional heat step required for activation like with other hot start technologies 4. Fidelity 6X greater than standard Taq 5. Generates 3:1 A-tailed to blunt ends 6. Functionally tested to prevent primer dimers and PCR amplify a 20.7 kb product Yes HotStart-IT Taq DNA Polymerase Hot start Taq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific PCR. Provided with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 1.25 units/µl 3. No additional heat step required for activation like with other hot start technologies 4. Functionally tested to prevent primer dimers in standard PCR 5' 3' No MagniTaq Multiplex PCR Master Mix (2X) Multiplex master mix with a chemically-modified hot start DNA polymerase for highly sensitive and specific simultaneous amplification of even the most difficult templates (including FFPE) with minimal optimization. 1. Concentration: 2X 2. Activation step: 5 minutes at 95 C 3. Advanced buffer formulation that includes dntps and MgCl 2 for minimal optimization requirements 4. Validated performance for simultaneous amplification of at least 20 targets up to 1 kb in length. 5' 3' No VeriQuest Taq DNA Polymerase Chemically-modified hot start Taq DNA polymerase that prevents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Provided with 10X PCR reaction buffer and MgCl 2 for optimization. 1. Source: Recombinant (E. coli) 2. Concentration: 5 units/µl 3. Activation step: 10 minutes at 95 C 4. Functionally tested in standard PCR 5 3 No End point PCR Taq DNA Polymerase (5 units/μl) Thermostable Taq DNA polymerase for routine PCR up to 6 kb. Includes 10X PCR reaction buffer and MgCl 2 for optimization units 250 units 1,000 units 5,000 units Taq PCR Master Mix, 2X Supplied as a convenient 2X pre-mixed formulation containing Taq DNA Polymerase, nucleotides, and reaction buffer optimized for a wide variety of PCR applications reactions 4

5 AMPLIFY End point PCR (continued) Taq Master Mix Plus Contains Taq PCR Master Mix, 25 mm MgCl 2 and PCR- Qualified H 2 O reactions Tth DNA Polymerase (5 units/μl) Suitable for PCR in the presence of Mg 2+ ions. It also has an intrinsic reverse transcriptase activity in the presence of Mn 2+ ions. The reverse transcription activity using RNA as a template to prepare cdna may be achieved at high temperatures, thereby eliminating problems related to secondary structure units 1,000 units RubyTaq DNA Polymerase (1 unit/μl) Contains high-quality Taq DNA Polymerase with two inert dyes that serve as tracking dyes in gel electrophoresis. Includes 10X PCR buffer and 25 mm MgCl 2 for optimization units 250 units 1,000 units 5,000 units RubyTaq PCR Master Mix (2X) Convenient 2X pre-mixed formulation containing Taq DNA Polymerase, nucleotides, reaction buffer, and two inert dyes reactions 500 reactions FideliTaq DNA Polymerase (5 units/μl) Combines high quality recombinant Taq DNA Polymerase with a high-fidelity, proofreading polymerase. Includes 10X PCR buffer and 25 mm MgCl 2 for optimization units 250 units 1,000 units 5 x 250 units 5,000 units FideliTaq PCR Master Mix, 2X Ready-to-use mix that combines Taq DNA Polymerase, a high-fidelity, proofreading polymerase, and nucleotides in a proprietary buffer formulation reactions FideliTaq PCR Master Mix Plus Includes FideliTaq PCR Master Mix (PN 71182) plus PCR-Qualified H 2 O and MgCl 2 solution reactions RubyTaq DNA Polymerase visualization during gel electrophoresis. RubyTaq Polymerase in PCR does not require the use of additional loading buffer. Simply load onto an agarose gel directly after cycling. During electrophoresis, RubyTaq separates into 2 colors, magenta (runs between 500 bp [2% gels] and 1500 bp [0.8% gels]) and yellow (runs less than 10 bp). The indicated volumes of RubyTaq DNA Polymerase were run on a 1% TAE agarose gel. 5

6 AMPLIFY End point direct PCR: Skip DNA extraction VersaTaq Direct PCR Polymerase (25 μl reaction volume) In contrast to standard Taq DNA polymerase, VersaTaq Direct PCR Polymerase can amplify DNA directly from samples, which eliminates costly extraction and purification steps, saving time and enabling amplification from limited quantities of DNA. Whole blood (up to 40%) Feces Blood spots on sample Urine collection cards Animal tissue Buccal swabs Plant tissue Soil (humic acid) Crude extracts reactions 200 reactions 500 reactions 2 x 500 reactions Mouse Tail (length) 2000 bp- 500 bp- M 1 mm M 1 2 Amplification of targets directly from mouse tail by VersaTaq Direct PCR Polymerase. Targets were amplified from 1 mm mouse tail clip added directly into PCR reaction. Lane 1: NUMB 483 bp; Lane 2: NUMB 1.8 kb. Blood (%) Anticoagulant E H C E H C E H C E H C E H C 2000 bp - M 500 bp - E: Na-EDTA H: Na-Heparin C: Na-Citrate M Amplification of targets directly from human whole blood. Targets were amplified from different concentrations of human whole blood in the presence of varying types of anticoagulants. Lanes 1-6: NUMB 455 bp; Lanes 7-12: NUMB 1.8 kb; Lanes 13-15: HRES1 488 bp - corresponding band is an 81% GC amplicon. 1 M betaine added to reactions with high GC targets. Amplicon GC % 48% 37% 81% 75% Humic acid (ng/µl) bp- M 500 bp- M Amplification of diverse targets in presence of humic acid by VersaTaq Direct PCR Polymerase. Targets were amplified from 5 ng of genomic DNA in the presence of varying concentrations of humic acid. Lanes 1-2: NUMB 455 bp; Lanes 3-4: NUMB 1.8 kb; Lanes 5-6: HRES1 488 bp; Lanes 7-8: SIM2 1.2 kb. 1 M betaine added to reactions with high GC content. 6

7 AMPLIFY End point hot start PCR HotStart-IT Taq DNA Polymerase (1.25 units/μl) Hot start Taq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific PCR. Includes 10X PCR buffer and 25 mm MgCl 2 for optimization units 250 units 1,000 units 5 x 250 units 5,000 units HotStart-IT Taq Master Mix, 2X Convenient 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase and nucleotides in a proprietary reaction buffer reactions 100 reactions 500 reactions HotStart-IT Binding Protein ( 10 mg/ml) Active component in primer sequestration technique. Sufficient for 200 x 25 μl reactions μg HotStart-IT FideliTaq DNA Polymerase (2.5 units/μl) Hot start FideliTaq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific, long and accurate PCR. Includes 10X PCR reaction buffer and 15 mm MgCl 2 for optimization units 250 units 1,000 units 5,000 units HotStart-IT FideliTaq PCR Master Mix, 2X Convenient formulation that contains HotStart-IT FideliTaq DNA Polymerase and nucleotides in a proprietary reaction buffer reactions 100 reactions 500 reactions VeriQuest Taq DNA Polymerase (5 units/μl) Chemically-modified hot start Taq DNA polymerase that prevents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Includes 10X PCR reaction buffer and MgCl 2 for optimization units 250 units 1,000 units 5,000 units PCR Reaction Preparation without Hot Start Polymerase primers with self-complementary region primers hybridize to each other at low temperatures polymerases are recruited to hybrid primer-dimers are produced PCR Failure PCR Reaction Preparation with USB Hot Start Method 5 3 Binding Proteins Polymerase primers with self-complementary region binding proteins interact with primers primers are sequestered at low temperatures prevents non-specific hybridization and extension denaturation step inactivates binding proteins primers bind to specific targets PCR Success HotStart-IT method: primer sequestration 7

8 AMPLIFY End point RT-PCR Enzymes: VeriScript Reverse Transcriptase (200 units/µl) This reverse transcriptase is an engineered version of M-MLV with reduced RNase H activity and increased thermostability, allowing for first-strand cdna synthesis with more full length cdnas, up to 12.5 kb. VeriScript Reverse Transcriptase is active up to 50 C, which offers greater sensitivity and consistent performance for a wide range of RT applications. Active over a wide temperature range. Recommended incubation temperature of 42 C, but incubation temperatures up to 50 C may be used for RNA templates with difficult regions containing secondary structure or GC-rich regions. Robust cdna synthesis is achieved with templates as long as 12.5 kb Compatible with oligo dt, random, or gene-specific priming Picogram level sensitivity in RT end point PCR Efficient amplification in RT real-time PCR from as few as 5 copies of synthetic RNA ,000 units 10,000 units 4 x 10,000 units 12.0 kb kb kb kb bp bp - M 0.5 kb 2.8 kb 6.6 kb M amplicon length T ( C) HeLa total RNA (1 µg) was used in a 20 µl first-strand cdna synthesis with 200 units of VeriScript Reverse Transcriptase. Reactions were incubated at the temperatures indicated on the figure for 30 minutes, followed by heat inactivation for 15 minutes at 70 C. RNA was removed by adding 1 µl (5 units) RNase H (PN 70054) and incubation at 37 C for 10 minutes, followed by heat inactivation for 10 minutes at 65 C. 10 ng cdna was used in a 50 µl PCR reaction using FideliTaq DNA Polymerase (PN 71180) for 35 cycles. VeriScript Reverse Transcriptase is active over a wide temperature range. Incubation temperatures up to 50 C may be used for RNA templates with difficult regions containing secondary structures or GC-rich regions. M-MLV Reverse Transcriptase (200 units/μl) This enzyme has a low RNase H activity that results in high yields of full length cdna ,000 units 100,000 units AMV Reverse Transcriptase (25 units/μl) Catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids; Synthesizes cdna for PCR, cloning and hybridization probes Y 200 units 70041Z 1,000 units One-step master mixes: RT-PCR Master Mix, 2X Includes M-MLV Reverse Transcriptase, Taq DNA Polymerase, recombinant RNase Inhibitor, nucleotides, and magnesium in a novel RT-PCR buffer reactions FideliTaq RT-PCR Master Mix, 2X Includes M-MLV Reverse Transcriptase, FideliTaq DNA Polymerase, recombinant RNase Inhibitor, nucleotides, and magnesium in a novel RT-PCR buffer reactions Kits: One-Step RT-PCR Kit The One-Step RT-PCR Kit is designed for simple RT-PCR in a one-tube format. Streamlines and optimizes the RT and PCR steps Provides a starting point for analysis of new RNA targets reactions Two-Step RT-PCR Kit The Two-Step RT-PCR Kit is designed for flexible RT-PCR in one- or two-tube formats. Optimization of PCR independent of RT Analysis of the expression of multiple genes in individual RNA samples with oligo dt primers RT reactions and 100 PCR reactions 8

9 AMPLIFY nucleotides dntps, Set of Four (da, dc, dg, dt), 100 mm Solutions 4 dntps per pack x 25 μmol (250 μl) dntps, Set of Four (da, dc, dg, dt), 100 mm Solutions 4 dntps per pack x 100 μmol dntps, Set of Four (da, dc, dg, dt), 100 mm Solutions 4 dntps per pack x 500 μmol PCR Nucleotide Mix, 10 mm Solution μl 2 x 500 μl PCR Nucleotide Mix, 25 mm Solution μl Multiplex PCR MagniTaq Multiplex PCR Master Mix, 2X Single-tube, 2X multiplex PCR master mix with a chemically-modified hot start DNA polymerase for highly sensitive and specific simultaneous amplification of at least 20 targets up to 1 kb in length of even the most difficult templates (including FFPE) with minimal optimization ng template DNA MagniTaq Multiplex PCR Master Mix, 2X contains hot start MagniTaq DNA Polymerase, dntps, and MgCl 2 in a proprietary buffer formulation designed to enable multiplex PCR without lengthy optimization procedures reactions 100 reactions µm primers: plex amplification of low amounts of gdna. 22-plex amplification of gdna targets with MagniTaq Multiplex PCR Master Mix. 1, 10, 100, and 1000 ng human genomic DNA input amounts (1 ng is about 100 cells) are added to the 22-plex notchpathway single-copy gene primer set. Primer concentration is 0.2 µm and samples were loaded with SYBR Green dye and run on an agarose gel. 22-plex of a wide range of primer concentrations. MagniTaq Multiplex PCR Master Mix is effective over a wide range of primer concentrations. 100 ng human genomic DNA input amount is added to the 22-plex notch-pathway, single-copy gene primer set. Duplicate reactions were run using 0.1, 0.2 or 0.4 µm primer concentrations. Samples were loaded with SYBR Green dye and run on an agarose gel. 9

10 AMPLIFY Real-time qpcr Instrument compatibility with USB VeriQuest qpcr Master Mixes Use this chart to determine which of our USB VeriQuest qpcr master mixes we recommend for your machine. Simply find your thermo cycler across the top row and then locate which of our master mixes are optimized for that instrument. VeriQuest SYBR Green qpcr Master Mix (2X) [75600] SYBR Green Mix Probe Mix Fast SYBR Green Mix Fast Probe Mix One-Step qrt-pcr SYBR Green Mix One-Step qrt-pcr Probe Mix VeriQuest SYBR Green qpcr Master Mix with Fluorescein (2X) [75665] VeriQuest Probe qpcr Master Mix (2X) [75650] VeriQuest Probe qpcr Master Mix, No Reference Dye (2X) [75660] VeriQuest Fast SYBR Green qpcr Master Mix (2X) [75690] VeriQuest Fast SYBR Green qpcr Master Mix with Fluorescein (2X) [75675] VeriQuest Fast Probe qpcr Master Mix (2X) [75680] VeriQuest Fast Probe qpcr Master Mix, No Reference Dye (2X) [75685] VeriQuest SYBR Green One- Step qrt-pcr Master Mix (2X) [75705] VeriQuest SYBR Green One-Step qrt-pcr Master Mix with Fluorescein (2X) [75715] VeriQuest Probe One-Step qrt-pcr Master Mix (2X) [75700] ABI Prism 7000 ABI Prism 7700 ABI Prism 7900 HT* ABI 5700 ABI 7300 ABI 7500* ABI StepOne /StepOne- Plus * ABI ViiA 7 Bio-Rad icycler iq /iq5 Bio-Rad icycler MyiQ Bio-Rad CFX96 /CFX384 Bio-Rad Opticon2 Bio-Rad Chromo4 Cepheid Smart Cycler Corbett Rotor-Gene Eppendorf Mastercycler ep realplex Fluidigm BioMark Illumina Eco */Helixis Pixo Qiagen Rotor-Gene Q* Roche Light Cycler 1.0, 1.5, 2.0* Roche LightCycler 480/1536* Stratagene Mx3000P * Stratagene Mx3005P * Stratagene Mx4000 TaKaRa TP-800 * Instruments can be set to fast mode cycling when using fast mode master mixes. Indicates preferred kit for this cycler Indicates may also be used for this cycler VeriQuest Probe One-Step qrt-pcr Master Mix, No Reference Dye (2X) [75710] VeriQuest qpcr solutions: Proven performance from a trusted source. Using a highly specific and sensitive master mix with a wide dynamic range is key for precise quantification and validation. VeriQuest qpcr reagents provide premium qpcr performance at affordable price points, allowing you to publish with confidence. Single-tube, 2X master mixes for SYBR green or TaqMan probe assays 4 C storage up to 4 months no need for freeze/thaw Performance on virtually any qpcr machine, fast cycling formulations available Superior consistency, sensitivity, and assay linearity VeriQuest Taq DNA Polymerase (5 units/μl) Chemically-modified hot start Taq DNA polymerase that prevents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Includes 10X PCR reaction buffer and MgCl 2 for optimization units 250 units 1,000 units 5,000 units 10

11 AMPLIFY Real-time qpcr (continued) For use with Applied Biosystems, Agilent, and Fluidigm instruments: VeriQuest SYBR Green qpcr Master Mix, 2X with ROX reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml) VeriQuest Probe qpcr Master Mix, 2X with ROX reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml) Linear detection range of VeriQuest SYBR Green qpcr Master Mix. Real-time amplification plot from a 10-fold dilution series of a GAPDH synthetic target with starting amounts of 10 9 copies amplified in four replicate reactions using the ABI StepOne Real-Time PCR System. References: 1. VanGuilder, H. D., Vrana, K. E., and Freeman, W. M. (2008) BioTechniques 44 (5), Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, Tewhey, R. et al. (2009) Nature Biotechnology 27, VeriQuest Fast SYBR Green qpcr Master Mix, 2X with ROX (20 μl reaction volume) reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml) Linear detection range of VeriQuest Probe qpcr Master Mix. Real-time amplification plot from a 10-fold dilution series of a GAPDH synthetic target with starting amounts of copies amplified in four replicate reactions using the ABI 7500 PCR System and GAPDH primer-probe set (FAM-BHQ -1). VeriQuest Fast Probe qpcr Master Mix, 2X with ROX (20 μl reaction volume) reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml) First-Strand cdna Synthesis Kit for Real-Time PCR Optimized for reverse transcription of RNA and produces first-strand cdna template suitable for real-time PCR reactions (20 μl) (Continued on next page) 11

12 AMPLIFY Real-time qpcr (continued) For use with BioRad, Eppendorf, and Roche instruments: VeriQuest SYBR Green qpcr Master Mix with Fluorescein, 2X reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml) VeriQuest Fast SYBR Green qpcr Master Mix with Fluorescein, 2X (20 μl reaction volume) reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml) VeriQuest Probe qpcr Master Mix, No Reference Dye, 2X reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml) VeriQuest Fast Probe qpcr Master Mix, No Reference Dye, 2X (20 μl reaction volume) reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml) ng RNA GAPDH Kras ng RNA Ct Kras y = x E PCR = 103.3% R² = GAPDH2 y = x E PCR = 97.7% R² = log input GAPDH2 Kras High sensitivity and precision in limited target quantification. Amplification plot (left) and standard curve (right) from realtime PCR for a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cdnas reverse-transcribed from HeLa total RNA. 12

13 AMPLIFY Real-time reverse transcription PCR (qrt-pcr) For use with Applied Biosystems, Agilent, and Fluidigm instruments: VeriQuest SYBR Green One-Step qrt-pcr Master Mix, 2X with ROX The 2X master mix contains 100X RT Enzyme Mix and 2X qrt-pcr Mix for RNA quantification with SYBR Green. The 100X RT Enzyme Mix is a blend of reverse transcriptase and RNase Inhibitor. The 2X qrt-pcr Mix contains hot start VeriQuest Taq DNA Polymerase, ultrapure nucleotides, SYBR Green I, and ROX Passive Reference Dye in an optimized buffer formulation for quantitative, real-time PCR detection with SYBR Green reactions (1 ml) 200 reactions (5 ml) VeriQuest Probe One-Step qrt-pcr Master Mix, 2X with ROX The 2X master mix contains reverse transcriptase, RNase inhibitor, our chemically modified VeriQuest Taq DNA polymerase, ROX Passive Reference Dye, and ultrapure nucleotides all in an optimized buffer composition reactions (1 ml) 200 reactions (5 ml) For use with BioRad, Eppendorf, and Roche instruments: VeriQuest SYBR Green One-Step qrt-pcr Master Mix with Fluorescein, 2X The 2X master mix contains 100X RT Enzyme Mix and 2X qrt-pcr Mix for RNA quantification with SYBR Green. The 100X RT Enzyme Mix is a blend of reverse transcriptase and RNase Inhibitor. The 2X qrt-pcr Mix contains chemically-modified VeriQuest Taq DNA Polymerase, ultrapure nucleotides, SYBR Green I, and Fluorescein Passive Reference Dye in an optimized buffer formulation for quantitative, real-time PCR detection with SYBR Green reactions (1 ml) 200 reactions (5 ml) VeriQuest Probe One-Step qrt-pcr Master Mix, No Reference Dye, 2X The 2X master mix contains reverse transcriptase, RNase inhibitor, our chemically modified VeriQuest Taq DNA Polymerase, and ultrapure nucleotides all in an optimized buffer composition reactions (1 ml) 200 reactions (5 ml) Exceptional results with performance comparisons. Ct ABI TaqMan Fast Virus 1-Step ABI TaqMan RNA-to-CT 1-Step VeriQuest One-Step Probe ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg HeLa Total RNA 13 slope E PCR R 2 ABI TaqMan Fast Virus 1-Step % ABI TaqMan RNA-to-CT 1-Step % VeriQuest One-Step Probe % Product comparison VeriQuest Probe One-Step qrt-pcr Master Mix. Ct values, R2, and PCR efficiency from real-time PCR for a 10-fold dilution series of 100 ng to 100 fg HeLa total RNA amplified in duplicate reactions with VeriQuest Probe One-Step qrt-pcr Master Mix, ABI TaqMan Fast Virus 1-Step Master Mix, and ABI TaqMan RNA-to-CT 1-Step Kit using the ABI 7500 Fast Real-Time PCR System and GAPDH primers and probe (Fam-BHQ) in standard mode with 15 minute RT reaction at 50 C.

14 PURIFY PCR product cleanup Which ExoSAP-IT reagent is right for you? ExoSAP-IT products offer a unique, one-tube, one-step, enzymatic process for PCR product cleanup. All ExoSAP-IT reagents provide 100% sample recovery with no loss of PCR products regardless of the fragment size. This PCR cleanup method removes excess primers and dntps and does not interfere with downstream applications. Use our comparison chart below to determine which formulation is best for your next experiment. ExoSAP-IT PCR Product Cleanup HT ExoSAP-IT High-Throughput PCR Product Cleanup HT ExoSAP-IT Fast High-Throughput PCR Product Cleanup Hands-on lab time Hands-off cleanup time 2 minutes 5 minutes (96 well) 3-5 minutes (96 well) 30 minutes 30 minutes 14 minutes Format Single tube Single tube 8-tube strips Single tube 8-tube strips Plates Throughput level Low to mid Recommended for processing 1-96 samples at a time High Recommended for processing 96 samples at a time High Recommended for processing 96 samples at a time Platform Single-channel pipette Single- or multi-channel pipette, robotic platform Single- or multi-channel pipette, robotic platform Cost per reaction Freezes at -20 C As low as $0.66 As low as $0.66 As low as $0.73 No Yes Yes Stability -20 C for up to 2 years. -20 C for up to 2 years. Once thawed, stable at 4 C for 1 week and RT for 8 hours. -20 C for up to 2 years. Once thawed, stable at 4 C for 1 month and RT for 2 days. 14

15 PURIFY PCR product cleanup (continued) HT ExoSAP-IT Fast High-Throughput PCR Product Cleanup From the leader in enzymatic PCR cleanup comes a new, faster option. Introducing HT ExoSAP-IT Fast for High-Throughput PCR Product Cleanup: a faster version of our flagship ExoSAP-IT reagent. Like the standard formulation, HT ExoSAP-IT Fast reagent is a mixture of Exonuclease I and Shrimp Alkaline Phosphatase for the enzymatic removal of excess primers and dntps following a PCR reaction. When PCR amplification is complete, any unconsumed dntps and primers remaining in the PCR product mixture will interfere with downstream applications. The ExoSAP-IT products remove these contaminants. HT ExoSAP-IT Fast reagent is scalable for PCR cleanup from a single tube up to a 384-well plate PCR reaction. Adding to our family of patented ExoSAP-IT products, HT ExoSAP-IT Fast reagent quickly provides accurate and consistent results in high-throughput applications. HT ExoSAP-IT Fast reagent is over 50% faster than standard HT ExoSAP-IT reagent with a 14 minute total protocol time. Add it directly to the PCR product and incubate at 37 C for just 7 minutes. After PCR treatment, inactivate it by simply heating to 80 C for another 7 minutes. This product is ideal for high volume labs that require quick sample turn around. With such a brief and simple protocol, proper sample cleanup never needs to be sacrificed due to processing or instrument time constraints. Ensure the highest quality Sanger sequencing results while keeping sample processing moving quickly and efficiently. PCR products cleaned up with HT ExoSAP-IT Fast reagent prior to automated sequencing displayed superior sequencing results compared to the untreated equivalent. Why HT ExoSAP-IT Fast reagent? Half the time of standard enzymatic protocols 100% recovery and only 5 μl of PCR product needed One simple pipetting step Formulated and formatted for automated platforms and multi-channel pipettes The most stable ExoSAP-IT reagent available HT ExoSAP-IT Fast High-Throughput PCR Product Cleanup overview (Continued on next page) 15

16 PURIFY PCR product cleanup (continued) High quality sequencing data after treatment with HT ExoSAP-IT Fast reagent HT ExoSAP-IT Fast reagent Treated Untreated Sequencing of a 1 kb PCR product cleaned with HT ExoSAP-IT Fast reagent (top) or untreated (bottom). HT ExoSAP-IT Fast treatment prior to sequencing eliminates miscalls and improves sequencing quality scores (numbers and bars above sequence; >50, probability of error 0.001). Sequence shown is within the first 150 bases. HT ExoSAP-IT Fast reagent displayed excellent stability in all formats tested including vials, 8-tube strips, and 96-well plates. The product showed no loss in function after 10 freeze thaw cycles, and is stable at variable temperatures for extended periods of time compared to enzyme mix equivalents. With excellent stability and a greater than 50% faster protocol, HT ExoSAP-IT Fast reagent is ideal for high-throughput assays where robotic platforms are utilized and large volumes of samples are processed in successive testing runs reactions 1 each 480 reactions x 8-tube strip 1 pack 5,760 reactions x 1 plate (12 x 8-tube strips) 4 pack 23,040 reactions x 4 plates 1,000 reactions 2 ml 5,000 reactions 10 ml 16

17 PURIFY PCR product cleanup (continued) HT ExoSAP-IT High-Throughput PCR Product Cleanup USB HT ExoSAP-IT High-Throughput reagent is an alternative formulation of ExoSAP-IT PCR Product Cleanup specifically designed for the unique requirements of highthroughput, automated platforms. HT ExoSAP-IT reagent has a decreased viscosity for robotic pipetting, yet maintains the same convenience and stability that you have come to expect from the ExoSAP-IT method. HT ExoSAP-IT High-Throughput PCR Cleanup reagent is designed to provide accurate and consistent results in high-throughput applications. High-throughput processing Even faster time to results with a low viscosity formulation allowing robotic pipetting Scalable Treat reaction volumes from 5 μl to 5 L Convenient packaging Available in 8-tube strips and 96-well formats High quality Accurate results In comparison to competitor products, HT ExoSAP-IT reagent allows for longer read lengths and greater confidence in the accuracy of those reads than did the alternative, AMPure XP beads. Sequencing data revealed that the bead-purified DNA had base miscalls, which is likely an effect of sample loss. HT ExoSAP-IT-treated samples showed no miscalls and had 100% recovery. HT ExoSAP-IT AMPure XP 3 miscalls Sequencing of a 1007 bp treated PCR product. A 1007 bp fragment was amplified and treated with HT ExoSAP-IT reagent (above) or Agencourt AMPure XP beads (below) and sequenced. Pherograms revealed no miscalls with HT ExoSAP-IT but three miscalls with Agencourt AMPure XP beads at position 203, 204, and reactions x 8-tube strip 5,760 reactions x 1 plate (12 x 8-tube strips) 23,040 reactions 4 plates 1,000 reactions 5,000 reactions 100% recovery from a range of fragment sizes with HT ExoSAP-IT reagent HT ExoSAP-IT reagent ensures 100% recovery and provides effective cleanup of all amplicon sizes. In contrast, AMPure XP beads were ineffective at purifying small amplicons whether determined by image analysis or by PicoGreen assay. Equivalent volumes of PCR product were visualized on an ethidium bromide agarose gel and the band volume determined using image analysis. HT-ExoSAP-IT reagent allowed recovery of 100% of the 86 and 103 bp PCR products but AMPure XP beads allowed only 8 and 14% recovery, respectively. Un Untreated, HT HT-ExoSAP-IT treated, XP AMPure XP purified, 100 bp Affymetrix 100 bp ladder, bands from 100 to 1000 bp by 100 bp intervals (PN 76712). 17 (Continued on next page)

18 PURIFY PCR product cleanup (continued) ExoSAP-IT PCR Product Cleanup Ideal cleanup for Sanger sequencing, TA cloning, in vitro transcription, and SNP analysis No spin columns! 100% recovery of DNA guaranteed Single pipette step, hands-off protocol Rapid PCR product cleanup protocol ExoSAP-IT reagent requires only one pipetting step and two incubations. Just add ExoSAP-IT reagent to the PCR product and within 30 minutes sequencing or SNP analysis can be performed. Simple: single-step The method is designed to require a minimum of handson time. Enzymatic removal of excess primers and unincorporated nucleotides occurs in one easy step by using ExoSAP-IT reagent in a single tube or microtiter well. Only simple pipette transfers are required, therefore, many samples can be processed at once, either manually or with robotics. No sample loss Use of ExoSAP-IT reagent eliminates all gel or column purifications, sedimentations, filtrations, beads, and/or magnetic separations (1). There is 100% recovery of both short and long PCR products with ExoSAP-IT reagent. 12 kb 2 kb 1 kb 100 bp HES-1 numb NRAGE numb M pre post pre post pre post pre post ExoSAP-IT treatment of PCR products with no sample loss. Single-copy targets were amplified from human genomic DNA. HES-1 (125 bp), numb (455 bp), NRAGE (1.55 kb), and numb (4.6 kb) were loaded on a 1.5% agarose gel before (pre) and after (post) ExoSAP-IT treatment. M is the DNA marker lane. Note that a variety of PCR product sizes can be treated with ExoSAP-IT, even a 125 bp fragment, with no sample loss. ExoSAP-IT PCR Product Cleanup overview 18

19 PURIFY PCR product cleanup (continued) USB ExoSAP-IT reagent USB ExoSAP-IT reagent GCATcGCCTGCTAAGCT-GCC GCATCGCCTGCTAAGCT-GCC Spin column Alternative enzymatic reagent GCAN-GCCNGCTAAGCTCGCC 2 miscalls Fluorescent sequencing results of a 100 bp puc18 PCR fragment sequenced with a -20 Fwd primer using fluorescent sequencing reagents. PCR cleanup performed with: (a) ExoSAP-IT; (b) a column designed for PCR cleanup. Base miscalls in (b) are due to inherently low yields of short PCR products when using columns reactions reactions reactions ,000 reactions ,000 reactions 4 miscalls Fluorescent sequencing results of a 1007 bp PCR product. A 1007 bp fragment was amplified and treated with ExoSAP-IT reagent (above) or an alternate enzymatic reagent (below) and sequenced. Pherograms revealed no miscalls with ExoSAP-IT reagent but four miscalls at position 25, 26, 30, and 39 with the alternate enzymatic reagent. An amount of 5 µl of PCR product was treated with 2 µl ExoSAP-IT reagent or the alternate enzymatic method per its protocol. Note: Two of the four miscalls are frame-shifts. References: 1. Dugan, K. A., Lawrence, H. S., Hares, D. R., Fisher, C. L. and Budowle B. (2002) J. Forensic Sci 47, Hanke, M. and Wink, M. (1994) BioTechniques 17, Mu, J., Duan, J., Makova, K., Joy, D., Huynh, C., Branch, O., Li, W. and Su, X. (2002) Nature 418, Silva, Jr., W. A., Costa, M. C. R., Valente, V., De Freitas Sousa, J., Paco- Larson, M. L., Espreafico, E. M., Camargo, S. S., Monteiro, E., De Jesus, A., Holanda, M. A., Zago, M. A., Simpson, A. J. G. and Neto, E. D. (2001) BioTechniques 30, Werle, E., Scneider C., Renner, M., Volker, M. and Fiehn, W. (1994) Nucl. Acids Res. 22,

20 PURIFY PCR purification PCR Product Pre-Sequencing Kit The PCR Product Pre-Sequencing Kit uses a novel enzymatic cleanup method to pre-treat PCR products prior to sequencing without any subsequent purification or separation steps. The two hydrolytic enzymes used in this kit, Shrimp Alkaline Phosphatase (SAP) and Exonuclease I, effectively remove excess dntps and primers present in the final PCR product reaction mixture. The enzymes are conveniently added to an aliquot of the PCR product mixture, incubated at 37 C, and then inactivated at 80 C. The result is a cleaner PCR product, free from excessive nucleotides and primers, ready to be sequenced with standard sequencing reagents reactions reactions ,000 reactions PrepEase PCR Purification 96-Well Plate Kits (Ultrafiltration) Designed for high-throughput purification of PCR products using 96-well plates x 96-well plates x 96-well plates Nucleic acid purification DNA isolation PrepEase DNA Cleanup Kits Binding capacity up to 15 μg Designed for the purification of DNA from reactions or for buffer exchange preps preps RNA purification PrepEase RNA Spin Kits Binding capacity up to 100 μg Designed for the isolation of total RNA from cells and tissue preps preps Plasmid purification PrepEase Plasmid Gravity-Flow Column Kits Designed to obtain low to high copy plasmid DNA from bacterial cultures. PrepEase Midi Plasmid Kit Binding capacity up to 100 μg Culture volume up to 30 ml preps PrepEase Maxi Plasmid Kit Binding capacity up to 500 μg Culture volume up to 150 ml preps PrepEase Quick MiniSpin Plasmid Kit Designed for very rapid purification preps PrepEase MiniSpin Plasmid Kit Designed for high yield purification preps PrepEase Gel Extraction Kits Binding capacity up to 15 μg Designed for purifying DNA fragments from agarose gels. Also suited for PCR product purification preps preps PrepEase Genomic DNA Isolation Kits Designed for the rapid isolation of genomic DNA preps preps 20

21 ANALYZE Next Generation sequencing products Prep2Seq DNA Library Prep Kit for Illumina platform Complete kit with the buffers and enzymes needed for high performing NGS libraries. The Prep2Seq DNA Library Prep Kit combines end repair and A-tailing steps, decreasing the total number of steps to achieve quality NGS library preparation. Only one cleanup step is required to attain peak performance on the Illumina sequencing platform and only 100 ng of input is needed reactions 20 reactions Prep2Seq Multiplex Oligo Adapters for Illumina platform Set 1 Contains adapter sequences KT Set 2 Contains adapter sequences KT Enzymes for Next Generation sequencing Key USB enzymes: Key features Relevance in ampliconbased NGS Relevance in adapter ligationbased NGS End repair and A-tailing Conversion of RNA to DNA Phosphorylation of DNA Adapter ligation MagniTaq Multiplex PCR Master Mix Simultaneous amplification of 20 targets from difficult templates ü Exonuclease-Free Klenow Klenow fragment lacking 3-5 exonuclease activity ü ü rdnase I, RNase-Free Free of RNases, sufficient removal of DNA contaminants ü ü ü T4 Gene 32 Protein Enhances cdna synthesis and T4 DNA Polymerase ü ü ü SSB, E. coli Stimulates DNA sequencing polymerase and enables longer read lengths ü ü Exonuclease I Removal of ssdna primers in between PCR amplifications, irreversible heat inactivation ü Exonuclease VII Only bi-directional exonuclease with single-stranded specificity, no requirement for divalent cations ü T4 Polynucleotide Kinase Phophorylation of nucleic acid 5 -ends and hydroxylation of 3 -ends. Use with OptiKinase for least bias and optimal sequencing coverage ü ü OptiKinase Reduces 5 end bias in kinase reactions and drives 3 phosphatase activity of T4 PNK for enhanced sequencing coverage ü ü T4 DNA Ligase, High Concentration Efficient adapter ligation to DNA fragments ü ü ü T4 RNA Ligase Efficient adapter ligation to RNA fragments ü ü NGS workflow solutions Prep2Seq DNA Library Prep Kit Complete kit with the buffers and enzymes needed for high performing NGS libraries on Illumina platform ü ü ü ü ExoSAP-IT PCR Product Cleanup Gold standard PCR cleanup for rapid sequencing of NGS drop-outs ü ü PrepEase Genomic DNA Isolation Kit Rapid isolation of gdna for NGS ü ü Change-IT Multiple Mutation Site Directed Mutagenesis Kit Efficient mutagenesis for gene function reverse genetics studies by NGS ü ü 21

22 ANALYZE Mutagenesis Change-IT Multiple Mutation Site Directed Mutagenesis Kit The Change-IT Multiple Mutation Site Directed Mutagenesis Kit is designed to create single or multiple oligonucleotide-directed sequence changes in plasmids. It also allows for efficient mutagenesis for gene function reverse genetics studies by NGS reactions Sequenase enzyme and kits Sequenase Version 2.0 DNA Polymerase Sequenase Version 2.0 is highly processive, incorporates nucleotide analogs (dltp, thio-dntps, dideoxy-ntps, etc.), is not impeded by secondary structures, and can carry out strand displacement synthesis. It is an excellent enzyme for dideoxy-sequencing and is useful in other applications, especially where the absence of associated exonuclease activity is desirable Y 200 units 70775Z 1,000 units Sequenase Version 2.0 DNA Sequencing Kit This kit features Sequenase Version 2.0 DNA Polymerase which is highly processive and able to effectively incorporate nucleotide analogs for sequencing. Use of the specially formulated buffers and mixes included in the kit will maximize yield of sequence information reactions Sequenase Quick Denature Plasmid DNA Sequencing Kit This kit offers two simple and efficient denaturation methods that do not require precipitation of plasmid DNA. Both methods, Glycol/Glycerol and Alkali/Acid, allow for rapid generation of denatured template DNA suitable for use with standard Sequenase Version 2.0 DNA Polymerase. In addition, this kit contains the 7-deaza-dGTP nucleotide mixes which provide stronger band intensities for resolving sequencing gel compressions reactions Thermostable sequencing enzymes and kits USB CycleSeq Thermostable DNA Polymerase (32 units/µl) USB CycleSeq Thermostable DNA Polymerase is a thermostable, DNA polymerase that is exonuclease-free and incorporates ddntps with a marked level of efficiency over standard thermostable DNA polymerases. This allows for more even and easy-to-read sequence band patterns, making sequence anomalies easier to identify. It is useful for cycle sequencing (Sanger sequencing), as it produces sequence data with uniform band intensities, allowing for longer and more accurate sequence reads. It is also useful for primer extension protocols during SNP genotyping ,000 units 10,000 units USB CycleSeq Polymerase sequencing results are comparable to GE Thermo Sequenase. Cycle sequencing using a fluorescently-labeled primer and 300 ng puc19 template DNA was performed using either Thermo Sequenase DNA Polymerase or CycleSeq Thermostable DNA Polymerase. Analysis of both sequences shows equivalent quality, read length, and band intensity. Thermo Sequenase Cycle Sequencing Kit This kit allows for equally efficient incorporation of both ddntps and dntps in cycle sequencing reactions resulting in very uniform band intensities reactions 22 Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit Kit is designed to be used with fluorescent dye-labeled primers and high-resolution fluorescence scanners reactions

23 ANALYZE Modifying enzymes AMV Reverse Transcriptase (15 units/μl) Catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids; Synthesizes cdna for PCR, cloning and hybridization probes Y 200 units 70041Z 1,000 units Calf Intestinal Alkaline Phosphatase (CIAP) (20 units/μl) Utilized for dephosphorylation of 5 -phosphorylated ends of DNA or RNA for subsequent labeling ,500 units Exonuclease I Effective for removal of ssdna primers in between PCR amplifications, irreversible heat inactivation. Standard concentration 10 units/μl 70073Z 2,500 units 70073X 5,000 units High concentration 20 units/μl ,000 units Exonuclease VII (10 units/μl) Only bi-directional exonuclease with single-stranded specificity, no requirement for divalent cations Y 200 units 70082Z 1,000 units Exonuclease-Free Klenow (10 units/μl) Klenow fragment lacking 3-5 exonuclease activity Y 125 units 70057Z 750 units ,500 units M-MLV Reverse Transcriptase (200 units/μl) This enzyme has a low RNase H activity that results in high yields of full length cdna ,000 units 100,000 units OptiKinase (10 units/μl) Reduces 5 end bias in kinase reactions and drives 3 phosphatase activity of T4 PNK for enhanced sequencing coverage X 500 units 78334Y 1,000 units 78334Z 2,500 units rdnase I, RNase-Free (10 units/μl) Free of RNases, sufficient removal of DNA contaminants ,000 units 2,500 units Shrimp Alkaline Phosphatase (SAP) (1 unit/μl) Used to treat unincorporated dntps in PCR reactions to prepare templates for DNA sequencing or SNP analysis units 1,000 units 5,000 units Single-stranded DNA Binding Protein (SSB) (5 μg/μl) SSB stimulates DNA sequencing polymerase and enables longer read lengths Y 100 μg 70032Z 500 μg T4 DNA Ligase Efficient adapter ligation to DNA fragments Standard concentration, 1 unit/μl 70005Y 100 units 70005X 500 units ,000 units High concentration, 10 units/μl 70042X 500 units ,000 units T4 Gene 32 Protein T4 Gene 32 Protein enhances cdna synthesis and T4 DNA Polymerase. Standard concentration, 5 μg/μl 70029Y 100 μg 70029Z 500 μg High concentration, 10 μg/μl 74029Y 300 μg 74029Z 1,000 μg Tth DNA Polymerase (5 units/μl) Suitable for PCR in the presence of Mg 2+ ions. It also has an intrinsic reverse transcriptase activity in the presence of Mn 2+ ions. The reverse transcription activity using RNA as a template to prepare cdna may be achieved at high temperatures, thereby eliminating problems related to secondary structure units 1,000 units (Continued on next page) 23

24 ANALYZE Modifying enzymes (continued) T4 Polynucleotide Kinase (30 units/μl) Phophorylation of nucleic acid 5 -ends and hydroxylation of 3 -ends. Use with OptiKinase for least bias and optimal sequencing coverage 70031Y 500 units 70031Z 1,000 units 70031X 2,500 units T4 RNA Ligase (10 units/μl) Efficient adapter ligation to RNA fragments units 2,500 units VeriScript Reverse Transcriptase (200 units/µl) This reverse transcriptase is an engineered version of M-MLV with reduced RNase H activity and increased thermostability, allowing for first-strand cdna synthesis with more full length cdnas, up to 12.5 kb. VeriScript Reverse Transcriptase is active up to 50 C which offers greater sensitivity and consistent performance for a wide range of RT applications ,000 units 10,000 units 4 x 10,000 units T4 RNA Ligase units 1,000 units PCR related reagents Agarose - Hi-Res, Separation 1000 bp gm 100 gm 500 gm Agarose - LE gm 100 gm 250 gm 500 gm 1 kg Agarose - Low Melt, Separation, 1000 bp Genetic Performance Certified gm 100 gm Agarose - Low Melt, Separation, 1000 bp Genetic Performance Certified gm 100 gm Agarose - Separation 500 bp Genetic Performance Certified gm 100 gm 250 gm 500 gm Betaine, 5 M Solution ml 5 x 1.5 ml 10 ml DNA Ladder, 1 kb Plus µl DNA Ladder, 100 bp µl Fluorescein Passive Reference Dye µl Ligate-IT Rapid Ligation Kit reactions reactions 24

25 ANALYZE PCR related reagents (continued) Magnesium Chloride (MgCl 2 ), 1 M Solution x 1 ml 100 ml Mineral Oil ml 25 ml 1 L PCR Markers, bp μl RapidRun Agarose Buffer, 20X Solution L 5 L ROX Passive Reference Dye μl Sodium Acetate (NaOAc) 3M Solution, ph ml 500 ml TAE Buffer, 10X Solution L 5 L TBE Buffer, 5X Solution L 5 L TE Buffer, 1X Solution x 1 ml 100 ml 500 ml TE Buffer, 50X Solution ml 500 ml Tris/HCl, 1M Solution, ph ml 500 ml 1 L Tris/HCl, 1M Solution, ph ml 500 ml 1 L Tris/HCl, 1M Solution, ph ml 500 ml 1 L Water, Nuclease-Free x 1 ml 100 ml 500 ml 1 L 5 L 25

26 OEM, custom, and bulk reagent solutions We provide custom and OEM solutions to meet your growing needs. USB has been helping diagnostic, biotech, and pharmaceutical companies succeed in the life sciences industry for over 40 years. We can confidently assist you with custom reagent manufacturing, dispensing, labeling, and kitting of your critical reagent components. Optimize your use of key staff and limited resources by outsourcing production of your finished goods or key components. Custom and OEM solutions: Enzymes, biochemicals and detergents Custom or bulk enzymes, formulated to your special requirements Special fill sizes/concentrations/formulations of over 3,000 catalog products OEM labeling of individual components, or complete kit packaging and labeling with your brand General Purpose Reagents (GPR) and IVD reagents and assays to your exacting standards Customer-specific quality control assays Our experts will work with you at your site and/or from our reagents center of excellence ISO compliant processes Rigorous quality system suited for your diagnostic requirements Comprehensive documentation Rigorous change control capabilities We welcome audits of our manufacturing facilities Get started today! Let us help you with your next project. Contact us at ( outside the US), or us at to get started. Please visit our website at usb.affymetrix.com for international distributor contact information. Product portfolio Here s a sampling of our more than 3,000 customizable products: Enzymes Ligases, kinases, phosphatases, polymerases, and Sequenase Convenience reagents Pre-mixed buffers and stock solutions PCR reagents ExoSAP-IT PCR Cleanup reagent for both standard and high throughput, SSB binding protein, DNA polymerases, and exo-free klenow Nucleotides dntps, PCR nucleotide mixes, ddntps, and NTPs Biochemicals Agaroses, acrylamides, amino acids, sugars, substrates, and vitamins 26