Premix Ex Taq (Probe qpcr)

For Research Use Premix Ex Taq (Probe qpcr) Product Manual

Table of Contents I. Description... 3 II. Principle... 4 III. Components... 5 IV. Materials Required but not Provided... 5 V. Storage... 5 VI. Precautions Before Use... 5 VII. Protocol... 6 1. General Overview of PCR Reaction Conditions...6 2. Protocol using Thermal Cycler Dice Real Time System II...6 3. Protocol using ABI PRISM 7000/7700, Applied Biosystems 7300 Real-Time PCR System, or StepOnePlus Real-Time PCR System...8 4. Protocol using Applied Biosystems 7500/7500 Fast Real-Time PCR System... 10 5. Protocol using LightCycler System... 12 6. Protocol using Smart Cycler II System... 14 7. Procedures when performing Real-Time RT-PCRs...16 VIII. Experimental Example...17 IX. Quality Control and Definition of Activity...17 X. Related Products...18 2 URL:http://www.takara-bio.com

I. Description Premix Ex Taq (Probe qpcr) is designed for real-time PCR (qpcr) detection with the TaqMan *1 probe. *2 This product is also suitable for high-speed PCRs. Premix Ex Taq allows accurate target quantification and detection over a broad dynamic range and makes it possible to conduct highly reproducible and reliable real-time PCR analyses. The product is supplied premixed at 2X concentration to facilitate easy preparation of reaction mixtures. The 2X premixed reagent also contains Tli RNase H, a heat-resistant RNase H, to minimize PCR inhibition by residual mrna in reactions with cdna as a template. A combination of TaKaRa Ex Taq HS, a hot start PCR enzyme that uses an anti-taq antibody, and a buffer optimized for real-time PCR results in excellent suppression of non-specific amplification, high amplification efficiency, and high detection sensitivity in real-time PCR analyses. Advantages: (1) This product allows rapid and accurate detection and quantitative gene expression analysis by real-time PCR. (2) The premixed reagent at 2X concentration makes pipetting easy. (3) Includes TaKaRa Ex Taq HS, which is designed for hot start PCRs. The buffer system has been optimized for real-time PCRs, offering excellent amplification efficiency and highly sensitive detection. (4) The 2X premixed reagent includes Tli RNase H, a heat-resistant RNase H, to minimize PCR inhibition by residual mrna in reactions using cdna template. Compatible apparatuses include: Thermal Cycler Dice Real Time System II (Cat. #TP900/TP960) Thermal Cycler Dice Real Time System Lite (Cat. #TP700/TP760) ABI PRISM *3 7000/7700, Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System and StepOnePlus Real-Time PCR System (Life Technologies) LightCycler (Roche Diagnostics) Smart Cycler System *4 /Smart Cycler II System *4 (Cepheid) Notes: * 1: * 2: * 3: * 4: TaqMan is a registered trademark of Roche Molecular Systems. The 5' nuclease process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. Purchase of the product does not provide a license to use this patented technology. For detections with SYBR Green I, use SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat. #RR420A) or SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A) premixed with SYBR Green I. SYBR is a registered trademark of Molecular Probes Inc. ABI PRISM is a registered tradmark of Applera Corporation. SmartCycler is a registered trademark of Cepheid Corporation. URL:http://www.takara-bio.com 3

II. Principle This product is used to perform PCR amplifications with TaKaRa Ex Taq HS. Signal from amplification of PCR products is monitored using a TaqMan probe. 1. PCR PCR enables amplification of specific target gene fragments from a minute amount of DNA. Through repeated cycles of DNA heat denaturation, primer annealing, and extension by DNA polymerase, PCR allows up to a million-fold amplification of the target gene fragments within a short time. For amplifications, this product uses the hot start PCR enzyme TaKaRa Ex Taq HS, which prevents non-specific amplifications. TaKaRa Ex Taq HS Polymerase prevents mispriming or formation of primer dimers during reaction mixture preparation or other pre-cycling steps and allows high-sensitivity detections. 2. Fluorescence Detection TaqMan probe technique Oligonucleotides modified by a 5 fluorophore (e.g., FAM) and a 3 quencher (e.g., TAMRA) are added to the reaction system. Under annealing conditions, the TaqMan probe hybridizes specifically to the template DNA. Fluorescence of the fluorophore is suppressed by the quencher. During the extension reaction, the 5 3 exonuclease activity of Taq DNA polymerase degrades the hybridized TaqMan probe, releasing quencher suppression and allowing fluorescence detection. 1) Heat denaturation Probe Primer Fluorophore F Q Quencher 2) Primer annealing/probe hybridization Polymerase F Hybridization Q 3) Extension F Q F Q 4 URL:http://www.takara-bio.com

III. Components (1) Premix Ex Taq (Probe qpcr) (2X conc.) *1 1 ml x 5 (2) ROX Reference Dye (50X conc.) *2 200 μl (3) ROX Reference Dye II (50X conc.) *2 200 μl *1: *2: Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, and Tli RNase H 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. IV. Materials Required but not Provided - Gene amplification system for real-time PCRs (authorized instruments) - Reaction tubes or plates designed specifically for this purpose - PCR primers - TaqMan probe for detection (licensed probe) - Sterile distilled water - Micropipettes and tips (sterile) V. Storage Store at 4 (stable for up to 6 months). Every precaution should be taken to avoid contamination. 1. This product is shipped frozen at -20. Store the product at 4 after receipt. Before use, gently invert tube to make sure reagent is completely dissolved and evenly mixed. 2. This product may be frozen at -20 for long term storage. Once thawed, it should be stored at 4 and used within 6 months. VI. Precautions Before Use This section describes precautions for using this product. Read before use. (1) Before use, make sure the reagent is evenly mixed by gently inverting the tube several times without creating bubbles; otherwise the reagent may not provide sufficient reactivity. Do not mix by vortexing. When stored frozen at -20, Premix Ex Taq (Probe qpcr) (2X conc.) may precipitate. To dissolve the precipitate completely, warm it by hand or let stand at room temperature briefly, followed by inverting the tube several times. Make sure reagent is evenly mixed before use. (2) Place reagent on ice immediately after it has thawed. (3) This product is not supplied with TaqMan probe. (4) Use fresh disposable tips to minimize potential cross-contamination between samples when preparing reaction mixtures or dispensing aliquots. URL:http://www.takara-bio.com 5

VII. Protocol 1. General Overview of PCR Reaction Conditions Initial denaturation Step Temperature Time Detection Comment Initial denaturation 95 30 sec. Off Generally, 95 for 30 sec. is sufficient for initial denaturation in most cases, even with difficult to denature templates such as circular plasmids and genomic DNAs. This procedure may be extended to 1-2 min. at 95 depending on template condition. Prolonged denaturation may inactivate the enzyme. Therefore, do not perform denaturation for more than 2 min. Shuttle PCR (2 step PCR) number of cycles: 30-45 cycles Step Temperature Time Detection Comment Denaturation 95 3-5 sec. Off Annealing/ extension 56-64 20-30 sec. (31, 34 sec.) * On Generally the amplification product size for real-time PCRs does not exceed 300 bp. Therefore, 95 for about 3-5 sec. is usually sufficient. When optimizing reaction conditions, evaluate results using annealing/ extension temperature in the range of 56-64. If poor reactivity occurs, increasing incubation time for this step may improve results. *: Some apparatuses by Life Technologies do not allow a detection-step setting of 30 sec. or shorter. ABI PRISM 7700 allows a setting of 30 sec. or longer. ABI PRISM 7000 and Applied Biosystems 7300 allow a setting of 31 sec. or longer. Applied Biosystems 7500 allows a setting of 34 sec. or longer. 2. Protocol using Thermal Cycler Dice Real Time System II 1. Prepare the PCR reaction mixture shown below. <Per reaction> Reagent Volume Final Conc. Premix Ex Taq (Probe qpcr) (2X conc.) PCR Forward Primer (10 μm) PCR Reverse Primer (10 μm) TaqMan Probe Template dh2o (sterile distilled water) Total 12.5 μl 0.5 μl 0.5 μl 1 μl 2 μl 8.5 μl 25 μl 1X 0.2 μm *1 0.2 μm *1 *2 *3 6 URL:http://www.takara-bio.com

*1: *2: *3: Final primer concentration of 0.2 μm is most likely to yield good results. However, should further optimization be required, try adjusting primer concentrations in the range of 0.1-1.0 μm. The probe concentration varies depending on the model of real-time PCR apparatus used and the fluorescent labeling dye of the probe. Refer to the instrument manual and the probe data sheet to determine the appropriate concentration. When using the Thermal Cycler Dice Real Time System II, use a final concentration in the range of 0.1-0.5 μm. Optimal template quantity depends on the target copy concentration of the template solution. Prepare and test serial dilutions to select an appropriate quantity. Use no more than 100 ng of DNA template. When using cdna as a template in RT-PCRs (RT reaction mixture), template solution volume should not exceed 10% of the PCR reaction mixture (e.g., no more than 2.5 μl cdna template solution for a 25 μl PCR reaction). 2. Start the reaction. The recommended protocol for PCR reactions is the shuttle PCR standard protocol shown below. You may set the annealing/extension time to between 20 and 30 sec., but first try 30 sec., which generally yields stable results. (Refer to General Overview of PCR Reaction Conditions on page 6.) Shuttle PCR Standard Protocol Hold (initial denaturation) Number of cycles: 1 95 30 sec. 2 Step PCR Number of cycles: 40 95 5 sec. 60 30 sec. Note: TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. The initial denaturation step should occur at 95 for 30 sec., which is generally sufficient for initial template denaturation. Enzyme activity decreases with longer heat treatment. Prolonged denaturation can affect amplification efficiency and quantification accuracy. 3. After the reaction is complete, assess the amplification curve and create a standard curve if a quantitative determination will be performed. Please refer to the instruction manual for your real time PCR instrument to read about analytical methods. URL:http://www.takara-bio.com 7

3. Protocol using ABI PRISM 7000/7700, Applied Biosystems 7300 Real-Time PCR System, or StepOnePlus Real-Time PCR System * Follow the procedures provided in the manual of the respective apparatus. 1. Prepare the PCR reaction mixture shown below. <Per reaction> Reagent Volume Volume Final Conc. Premix Ex Taq (Probe qpcr) (2X conc.) PCR Forward Primer (10 μm) PCR Reverse Primer (10 μm) TaqMan Probe ROX Reference Dye (50X) *3 Template dh2o (sterile distilled water) 10 μl 0.4 μl 0.4 μl 0.8 μl 0.4 μl 2 μl 6 μl 25 μl 1 μl 1 μl 2 μl 1 μl 4 μl 16 μl Total 20 μl *5 50 μl *5 1X 0.2 μm *1 0.2 μm *1 *2 1X *4 *1: *2: Final primer concentration of 0.2 μm is most likely to yield good results. However, should further optimization be required, try adjusting primer concentrations in the range of 0.1-1.0 μm. The probe concentration varies depending on the model of real-time PCR apparatus used and the fluorescent labeling dye of the probe. Refer to the instrument manual and the probe data sheet to determine the appropriate concentration. *3: Use the ROX Reference Dye at a final concentration of 1X. *4: *5: Optimal template quantity depends on the target copy concentration of the template solution. Prepare and test serial dilutions to select an appropriate quantity. Use no more than 100 ng of DNA template. When using cdna as a template in RT-PCRs (RT reaction mixture), template solution volume should not exceed 10% of the PCR reaction mixture (e.g., no more than 2 μl cdna template solution for a 20 μl PCR reaction). Adjust according to the recommended volume for each apparatus. 2. Start the reaction. The recommended protocol for PCR reactions is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. (Refer to "General Overview of PCR Reaction Conditions on page 6.) Note: TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. The initial denaturation step should occur at 95 for 30 sec., which is generally sufficient for initial template denaturation. Enzyme activity decreases with longer heat treatment. Prolonged denaturation can affect amplification efficiency and quantification accuracy. 8 URL:http://www.takara-bio.com

< ABI PRISM 7000/7700, 7300 Real-Time PCR System > Shuttle PCR Standard Protocol Stage 1: initial denaturation Number of cycles: 1 95 30 sec. Stage 2: PCR Number of cycles: 40 95 5 sec. 60 30-31 sec. * *: Set the reaction time to 30 sec. for Models 7700 31 sec. for Models 7000 and 7300. < StepOnePlus Real-Time PCR System > Fast Protocol Holding Stage Number of cycles: 1 95 20 sec. Cycling Stage Number of Cycles: 40 95 1 sec. 60 20 sec. 3. After the reaction is complete, assess the amplification curve and create a standard curve if a quantitative determination will be performed. Please refer to the instruction manual for your real time PCR instrument to read about analytical methods. URL:http://www.takara-bio.com 9

4. Protocol using Applied Biosystems 7500/7500 Fast Real-Time PCR System * Follow the procedures provided in the manual of the respective apparatus. 1. Prepare the PCR reaction mixture shown. <Per reaction> Reagent Volume Volume Final Conc. Premix Ex Taq (Probe qpcr) (2X conc.) PCR Forward Primer (10 μm) PCR Reverse Primer (10 μm) TaqMan Probe ROX Reference Dye II (50X) *3 Template dh2o (sterile distilled water) 10 μl 0.4 μl 0.4 μl 0.8 μl 0.2 μl 2 μl 6.2 μl 25 μl 1 μl 1 μl 2 μl 0.5 μl 4 μl 16.5 μl Total 20 μl *5 50 μl *5 1X 0.2 μm *1 0.2 μm *1 *2 0.5X *4 *1: *2: *3: *4: *5: Final primer concentration of 0.2 μm is most likely to yield good results. However, should further optimization be required, try adjusting primer concentrations in the range of 0.1-1.0 μm. The probe concentration varies depending on the model of real-time PCR apparatus used and the fluorescent labeling dye of the probe. Refer to the instrument manual and the probe data sheet to determine the appropriate concentration. Use the ROX Reference Dye II at a final concentration of 0.5X. Optimal template quantity depends on the target copy concentration of the template solution. Prepare and test serial dilutions to select an appropriate quantity. Use no more than 100 ng of DNA template. When using cdna as a template in RT-PCRs (RT reaction mixture), template solution volume should not exceed 10% of the PCR reaction mixture (e.g., no more than 2 μl cdna template solution for a 20 μl PCR reaction). Adjust according to the recommended volume for each apparatus. 2. Start the reaction. The recommended protocol for PCR reactions is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. (Refer to "General Overview of PCR Reaction Conditions on page 6.) Note: TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. The initial denaturation step should occur at 95 for 30 sec., which is generally sufficient for initial template denaturation. Enzyme activity decreases with longer heat treatment. Prolonged denaturation can affect amplification efficiency and quantification accuracy. 10 URL:http://www.takara-bio.com

< 7500 Real-Time PCR System > Shuttle PCR Standard Protocol Stage 1: initial denaturation Number of cycles: 1 95 30 sec. Stage 2: PCR reaction Number of cycles: 40 95 5 sec. 60 34 sec. < 7500 Fast Real-Time PCR System > Fast Protocol Holding Stage Number of cycles: 1 95 20 sec. Cycling Stage Number of cycles: 40 95 3 sec. 60 30 sec. 3. After the reaction is complete, assess the amplification curve and create a standard curve if a quantitative determination will be performed. Please refer to the instruction manual for your real time PCR instrument to read about analytical methods. URL:http://www.takara-bio.com 11

5. Protocol using LightCycler System * Follow the procedures provided in the manual for LightCycler System (Roche Diagnostics). 1. Prepare the PCR reaction mixture shown below. <Per reaction> Reagent Volume Final Conc. Premix Ex Taq (Probe qpcr) (2X conc.) PCR Forward Primer (10 μm) PCR Reverse Primer (10 μm) TaqMan Probe Template dh2o (sterile distilled water) Total 10 μl 0.4 μl 0.4 μl 0.8 μl 2 μl 6.4 μl 20 μl 1X 0.2 μm *1 0.2 μm *1 *2 *3 *1: *2: *3: Final primer concentration of 0.2 μm is most likely to yield good results. However, should further optimization be required, try adjusting primer concentrations in the range of 0.1-1.0 μm. The probe concentration varies depending on the model of real-time PCR apparatus used and the fluorescent labeling dye of the probe. Refer to the instrument manual and the probe data sheet to determine the appropriate concentration. Optimal template quantity depends on the target copy concentration of the template solution. Prepare and test serial dilutions to select an appropriate quantity. Use no more than 100 ng of DNA template. When using cdna as a template in RT-PCRs (RT reaction mixture), template solution volume should not exceed 10% of the PCR reaction mixture (e.g., no more than 2 μl cdna template solution for a 20 μl PCR reaction). 12 URL:http://www.takara-bio.com

2. Briefly centrifuge PCR capillaries. Place them in the LightCycler instrument and start the reaction. The recommended protocol for PCR reactions is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. (Refer to "General Overview of PCR Reaction Conditions on page 6.) Note: TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. The initial denaturation step should occur at 95 for 30 sec., which is generally sufficient for initial template denaturation. Enzyme activity decreases with longer heat treatment. Prolonged denaturation can affect amplification efficiency and quantification accuracy. Shuttle PCR Standard Protocol Stage 1: initial denaturation 95 30 sec. 20 /sec. 1 Cycle Stage 2: PCR reaction 95 5 sec. 20 /sec. 60 20 sec. 20 /sec. 40 Cycles 3. After the reaction is complete, assess the amplification curve and create a standard curve if a quantitative determination will be performed. Please refer to the instruction manual for your real time PCR instrument to read about analytical methods. URL:http://www.takara-bio.com 13

6. Protocol using Smart Cycler II System 1. Prepare the PCR reaction mixture shown below. <Per reaction> Reagent Volume Final Conc. Premix Ex Taq (Probe qpcr) (2X conc.) PCR Forward Primer (10 μm) PCR Reverse Primer (10 μm) TaqMan Probe Template dh2o (sterile distilled water) Total 12.5 μl 0.5 μl 0.5 μl 1 μl 2 μl 8.5 μl 25 μl 1X 0.2 μm *1 0.2 μm *1 *2 *3 *1: *2: *3: Final primer concentration of 0.2 μm is most likely to yield good results. However, should further optimization be required, try adjusting primer concentrations in the range of 0.1-1.0 μm. The probe concentration varies depending on the model of real-time PCR apparatus used and the fluorescent labeling dye of the probe. Refer to the instrument manual and the probe data sheet to determine the appropriate concentration. When using the Smart Cycler System/Smart Cycler II System, generally, try a final concentration in the range of 0.1-0.5 μm. Optimal template quantity depends on the target copy concentration of the template solution. Prepare and test serial dilutions to select an appropriate quantity. Use no more than 100 ng of DNA template. When using cdna as a template in RT-PCRs (RT reaction mixture), template solution volume should not exceed 10% of the PCR reaction mixture (e.g., no more than 2.5 μl cdna template solution for a 25 μl PCR reaction). 14 URL:http://www.takara-bio.com

2. Briefly centrifuge the reaction tubes with a Smart Cycler centrifuge. Place them in Smart Cycler and start the reaction. The recommended protocol for PCR reactions is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. (Refer to "General Overview of PCR Reaction Conditions on page 6.) Note: TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. The initial denaturation step should occur at 95 for 30 sec., which is generally sufficient for initial template denaturation. Enzyme activity decreases with longer heat treatment. Prolonged denaturation can affect amplification efficiency and quantification accuracy. Shuttle PCR Standard Protocol Stage 1: initial denaturation Hold 95 30 sec. Stage 2: PCR reaction Repeat 40 times 95 5 sec. 60 20 sec. 3. After the reaction is complete, assess the amplification curve and create a standard curve if a quantitative determination will be performed. Please refer to the instruction manual for your real time PCR instrument to read about analytical methods. URL:http://www.takara-bio.com 15

7. Procedures when performing Real Time RT-PCRs For 2 step RT-PCRs, use in combination with PrimeScript RT reagent Kit (Perfect Real Time) (Cat. #RR037A - not available in the U.S.) is recommended. Perform the reverse transcription reaction using the TaqMan probe assay protocol below. 1. Prepare the reverse transcription reaction mixture shown below. Assemble the reaction mixture on ice. <Per reaction> Reagent Volume Final Conc. 5X PrimeScript Buffer (for Real Time) PrimeScript RT Enzyme Mix I Oligo dt Primer (50 μm) *1 Random 6 mers (100 μm) *1 total RNA RNase Free dh2o 2 μl 0.5 μl 0.5 μl 2 μl Total 10 μl *2 1X 25 pmol 200 pmol *1: Using both the Oligo dt Primer and the Random 6 mers allows efficient cdna synthesis for the entire region of mrna. The amount to use when using each primer alone or when using Gene Specific Primer is shown below. Primer Volume Amount Oligo dt Primer (50 μm) Random 6 mers (100 μm) Gene Specific Primer (2 μm) 0.5 μl 2 μl 0.5 μl 25 pmol 200 pmol 1 pmol *2: Scale up the reverse transcription reaction as necessary. A 10 μl reaction mixture volume can reverse-transcribe up to approximately 1 μg of total RNA. 2. Perform the reverse transcription reaction. 37 15 min. * (reverse transcription) 85 5 sec. (heat inactivation of reverse transcriptase) 4 *: When using the Gene Specific Primer: Perform reverse transcription at 42 for 15 min. If non-specific PCR amplification occurs, conducting the reverse transcription step at 50 may improve results. 3. Perform PCR. Perform a PCR according to the method described in VII. Protocol on page 6. 16 URL:http://www.takara-bio.com

VIII. Experimental Example Example (with Applied Biosystems 7500 Fast Real-Time PCR System) A real-time RT-PCR was performed to detect mouse Gapdh mrna with a primer supplied with TaqMan Gene Expression Assays (Life Technologies) and a TaqMan probe. Templates used were serial dilutions of cdna equivalent to between 1 pg and 100 ng of total RNA. A negative control contained dh2o in place of template. IX. Quality Control and Definition of Activity Quality test Real-time PCR reactions by Smart Cycler System with λdna as a template (amplification product size 300 bp) have yielded excellent and stable amplifications. TaKaRa Ex Taq HS Definition of activity Each 1 U is defined as the activity of incorporating 10 nmol of total nucleotides into acid-insoluble precipitate over 30 min. at 74 in the activity assay reaction mixture described below, using activated salmon sperm DNA as a template/primer. Composition of the reaction mixture for assaying activity 25 mm TAPS Buffer (ph9.3, 25 ) 50 mm KCl 2 mm MgCl2 0.1 mm DTT 200 μm each datp, dgtp, dctp 100 μm [ 3 H]-dTTP 0.25 mg/ml Activated Salmon Sperm DNA Purity 1. After 10 U of this enzyme and 0.6 μg of λ-hind III degradation product were incubated at 74 for 1 hour, no change in the DNA electrophoresis pattern was observed. 2. After 10 U of this enzyme and 0.6 μg of supercoiled pbr322 DNA were incubated at 74 for 1 hour, no change in the DNA electrophoresis pattern was observed. 3. After 10 U of this enzyme and 0.6 μg of λdna incubated at 74 for 1 hour, no change in the DNA electrophoresis pattern was observed. URL:http://www.takara-bio.com 17

X. Related Products PrimeScript RT reagent Kit (Perfect Real Time) (Cat #RR037A/B) PrimeScript RT reagent Kit with gdna Eraser (Perfect Real Time) (Cat #RR047A/B) PrimeScript RT Master Mix (Perfect Real Time) (Cat #RR036A/B) One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Cat #RR064A) SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat #RR820A/B) SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat #RR420A/B) One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (Cat #RR086A/B) Thermal Cycler Dice Real Time System II (Cat #TP900/TP960) * Thermal Cycler Dice Real Time System Lite (Cat #TP700/TP760) * *: Not available in all geographic locations. Check for availability in your area. 18 URL:http://www.takara-bio.com

NOTICE TO PURCHASER: LIMITED LICENSE [P7] PCR Notice A license to perform the patented 5' Nuclease Process for research is obtained by the purchase of (i) both Authorized 5' Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5' Nuclease Kit, or (iii) license rights from Applied Biosystems. This product is an Authorized 5' Nuclease Core Kit. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711, 6,127,155, 5,677,152 and 5,773,258. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. Separate purchase of a Licensed Probe would convey rights under the applicable claims of US Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569 and 5,804,375 (claims 1-12 only) and corresponding claims outside the United States. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [L52] Rox Reference Dye (Research Field) Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,928,907. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [M40] Thermostable RNaseH This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims. [M57] LA Technology This product is covered by the claims 6-16 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims. [M82] Tli RNaseH Plus This product is the subject of the pending JP patent application. Trademarks TaqMan is a registered trademark of Roche Molecular Systems. SYBR is a registered trademark of Molecular Probes Inc. Cycleave is a registered trademark of Takara Bio Inc. SmartCycler is a registered trademark of Cepheid Corporation. LightCycler is a registered trademark of Roche Diagnostics. ABI PRISM is a registered trademark of Applera Corporation. Other brand names and product names, unless otherwise specified, are also proprietary trademarks or registered trademarks. URL:http://www.takara-bio.com 19

NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at +81 77 543 7247 or from our website at www.takara-bio.com. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. 20 URL:http://www.takara-bio.com