SYBR Premix Ex Taq (Tli RNaseH Plus), Bulk

Transcription

1 Cat. # RR420L For Research Use SYBR Premix Ex Taq (Tli RNaseH Plus), Bulk Product Manual

2 Table of Contents I. Description... 3 II. Principle... 3 III. Kit Components... 4 IV. Materials Required but not Provided... 4 V. Storage... 4 VI. Precautions before Use... 5 VII. Protocol... 6 VIII. Appendix...13 IX. Related Products URL:

3 I. Description SYBR Premix Ex Taq (Tli RNaseH Plus) is a kit specifically designed for intercalator-based real-time PCR using SYBR Green I *1. It is supplied at a 2 concentration premixed with SYBR Green I at a concentration appropriate for real-time monitoring, making it easy to prepare reaction mixtures. 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 allows high amplification efficiency and high detection sensitivity in real-time PCR. The 2 premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes inhibition of PCR due to residual mrna when using cdna as template. This product is suitable for high-speed PCR and allows accurate assay and detection of targets, making it possible to perform real-time PCR analyses with good reproducibility and high reliability. Compatible instrument systems include: Thermal Cycler Dice Real Time System(Cat. # TP850/TP870) *2 Smart Cycler II System *3 (Cat. # SC200N/SC210N) Applied Biosystems 7500 Real-Time PCR System, Applied Biosystems 7500 Fast Real-Time PCR System, StepOnePlus Real-Time PCR System(Life Technologies) LightCycler /LightCycler 480 System(Roche Diagnostics) CFX96 Real-Time PCR Detection System (Bio-Rad) *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. *2: When using Thermal Cycler Dice Real Time System II (Cat. #TP900/TP960), the use of SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820L) is recommended. *3: Smart Cycler is a registered trademark of Cepheid Corporation. II. Principle SYBR Premix Ex Taq (Tli RNaseH Plus) is used for PCR amplifications with TaKaRa Ex Taq HS. PCR amplification products can be monitored in real time by SYBR Green I. TaKaRa Ex Taq HS, a hot start PCR enzyme, prevents non-specific amplifications derived from mispriming or primer dimer formation during reaction mixture preparation or other precycling steps and thereby makes high-sensitivity detection possible. Fluorescence detection intercalator method This method involves the addition of a reagent (intercalator: e.g., SYBR Green I) that emits fluorescence when bound to double-strand DNA in reaction mixtures, allowing the detection of fluorescence associated with amplification. Measuring the fluorescence intensity not only allows quantitative determination but also provides the melting temperature of amplified DNA. URL: 3

4 lii.kit Components (200 reactions, 50-µl volume) (1) SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk *1 5 ml 1 (2) ROX Reference Dye (50 ) *2 200 µl *1: Contains TaKaRa Ex Taq HS, dntp Mixture, Mg 2+, Tli RNaseH, and SYBR Green I *2: Use when performing analyses with an instrument that normalizes fluorescent signals between wells (e.g., real-time PCR systems from Life Technologies). The amount to use varies depending on the instrument system used. Add ROX Reference Dye (50 ) in a volume equivalent to 1/50 of the PCR reaction mixture when using the following instrument: StepOnePlus Real-Time PCR System (Life Technologies) Add ROX Reference Dye (50 ) in a volume equivalent to 1/250 of the PCR reaction mixture when using the following instrument: Applied Biosystems 7500/7500 Fast Real Time PCR System (Life Technologies) No ROX Reference Dye (50 ) is required when using any of the following instruments: Thermal Cycler Dice Real Time System(Cat. # TP850/TP870) Smart Cycler II System(Cat. # SC200N/SC210N) LightCycler /LightCycler 480 System(Roche Diagnostics) CFX96 Real-Time PCR Detection System(Bio-Rad) IV.Materials Required but not Provided - Gene amplification system for real-time PCR (authorized instruments) - Reaction tubes and plates designed specifically for the qpcr instrument used - PCR primers* - Sterile distilled water - Micropipettes and tips (sterile, with filter) *: For designing real time PCR primers, please see Section VIII. (2) Primer design. V.Storage Store at 4ºC (stable for up to 6 months). Protect this kit from light and avoid contamination. This kit is shipped frozen at 80 C. For long-term storage, keep at 80 C. (Do not store at 20 C.) Once thawed, it should be stored at 4 C and used within 6 months. 4 URL:

5 VI.Precautions before Use Read these precautions before use and follow them when using this product. 1. Before use, make sure the reagent is evenly mixed by inverting the tube several times without creating bubbles. Uneven reagent composition will result in inadequate reactivity. Do not mix by vortexing. When stored at 80ºC, SYBR Premix Ex Taq (2 ) (Tli RNase H Plus), Bulk may develop a white to pale yellow precipitate.to dissolve the precipitate completely, let stand briefly protected from light at room temperature (below approximately 30ºC), followed by inverting the tube several times. The presence of precipitate is indicative of uneven reagent composition; make sure the reagent is evenly mixed before use. 2. Place reagents on ice immediately after they have thawed. 3. This product contains SYBR Green I. Take care to avoid exposures to strong light when preparing the reaction mixture. 4. Use fresh disposable tips to avoid any potential contamination between samples when preparing or dispensing reaction mixtures. 5. TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-taq antibody that inhibits polymerase activity. Do not perform the pre-pcr incubation (5 to 15 minutes at 95 C) that is required with other companies chemically modified hot start PCR enzymes. The activity of TaKaRa Ex Taq HS decreases with longer heat treatment and the amplification efficiency and quantification accuracy can be affected. Even for the initial denaturation step, 95 C for 30 sec. is generally sufficient. URL: 5

6 VII. Protocol <For Thermal Cycler Dice Real Time System> 1. Prepare the PCR mixture shown below. (Per reaction) Reagent Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 12.5 µl 1 PCR Forward Primer(10 µm) 0.5 µl 0.2 µm *1 PCR Reverse Primer(10 µm) 0.5 µl 0.2 µm *1 Template (<100 ng) *2 2 µl dh2o (sterile distilled water) 9.5 µl Total 25 µl *3 *1: A final primer concentration of 0.2 µm is most likely to yield a good result. Nevertheless, if there is an issue with reactivity, try to find an optimal concentration between 0.1 and 1.0 µm. *2: The optimal quantity depends on the number of target copies in the template solution. Make serial dilutions to determine the appropriate amount. It is preferable to use no more than 100 ng of DNA template. Furthermore, if cdna (RT reaction mixture) is added as template, the template volume should be no more than 10% of the PCR mixture. *3: The recommended volume for reaction mixtures is 25 µl. 2. Start the reaction. The recommended protocol for PCR is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. Perform a 3-step PCR when using a primer with low T m value or when shuttle PCR is not feasible. Shuttle PCR standard protocol Hold (Initial denaturation) Cycle: sec. 2 Step PCR *4 Cycle: sec sec. Dissociation *4: For optimizing PCR conditions, please see VIII. (1) Optimization. 3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if an assay will be performed. For analytical methods, refer to the manual of Thermal Cycler Dice Real Time System. 6 URL:

7 <For Applied Biosystems 7500/7500 Fast Real-Time PCR System or StepOnePlus Real-Time PCR System> * Please follow the procedures provided in the instruction manual of the respective Life Technologies instrument system. 1. Prepare the PCR mixture shown below. Add ROX Reference Dye (50 ) in a volume equivalent to 1/50 of the PCR reaction mixture when using the StepOnePlus Real-Time PCR System. (Per reaction) Reagent Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 10 µl 1 PCR Forward Primer (10 µm) 0.4 µl 0.2 µm *1 PCR Reverse Primer (10 µm) 0.4 µl 0.2 µm *1 ROX Reference Dye (50 ) 0.4 µl 1 Template (< 100 ng) *2 2 µl dh2o (sterile distilled water) 6.8 µl Total 20 µl *3 Add ROX Reference Dye (50 ) in a volume equivalent to 1/250 of the PCR reaction mixture when using the Applied Biosystems 7500/7500 Fast Real-Time PCR System. (Per reaction) Reagent Volume Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 10 µl 25 µl 1 PCR Forward Primer (10 µm) 0.4 µl 1 µl 0.2 µm *1 PCR Reverse Primer (10 µm) 0.4 µl 1 µl 0.2 µm *1 ROX Reference Dye (50 ) 0.08 µl 0.2 µl 0.2 Template (< 100 ng) *2 2 µl 4 µl dh2o (sterile distilled water) 7.12 µl 18.8 µl Total 20 µl *3 50 µl *3 *1: A final primer concentration of 0.2 µm is most likely to yield a good result. Nevertheless, if there is an issue with reactivity, try to find an optimal concentration between 0.1 and 1.0 µm. *2: The optimal quantity depends on the number of target copies in the template solution. Make serial dilutions to determine the appropriate amount. It is preferable to use no more than 100 ng of DNA template. Furthermore, if cdna (RT reaction mixture) is added as template, the template volume should be no more than 10% of the PCR mixture. *3: Prepare in accordance with the recommended volume for each instrument. 2. Start the reaction. The recommended protocol for PCR is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. Perform a 3-step PCR when using a primer with low T m value or when a shuttle PCR is not feasible. For optimizing PCR conditions, please refer to Section VIII (1). URL: 7

8 <StepOnePlus Real-Time PCR System> Shuttle PCR standard protocol Holding Stage Step1: sec. Cycling Stage Number of Cycles: 40 Step1: 95 5 sec. Step2: sec. Melt Curve Stage <Applied Biosystems 7500 Real-Time PCR System> Shuttle PCR standard protocol Stage 1: Initial denaturation Number of cycles: sec. Stage 2: PCR Number of cycles: sec sec. Stage 3: Melt Curve <Applied Biosystems 7500 Fast Real-Time PCR System> Shuttle PCR standard protocol Holding Stage Step 1: sec. Cycling Stage Number of Cycles: 40 Step 1: 95 3 sec. Step 2: sec. Melt Curve Stage 3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if an assay will be performed. Refer to the instrument manual for analytical methods. 8 URL:

9 <For LightCycler /LightCycler 480 System> * Please follow the procedures provided in the manual for the respective LightCycler instrument from Roche Diagnostics. 1. Prepare the PCR mixture shown below. (Per reaction) Reagent Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 10 µl 1 PCR Forward Primer (10 µm) 0.4 µl 0.2 µm *1 PCR Reverse Primer (10 µm) 0.4 µl 0.2 µm *1 Template (<100 ng) *2 2 µl dh2o (sterile distilled water) 7.2 µl Total 20 µl *1: A final primer concentration of 0.2 µm is most likely to yield a good result. Nevertheless, if there is an issue with reactivity, try to find an optimal concentration between 0.1 and 1.0 µm. *2: The optimal quantity depends on the number of target copies in the template solution. Make serial dilutions to determine the appropriate amount. It is preferable to use no more than 100 ng of DNA template. Furthermore, if cdna (RT reaction mixture) is added as template, the template volume should be no more than 10% of the PCR mixture. 2. Start the reaction. The recommended protocol for PCR is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. Perform a 3-step PCR when using a primer with low T m value or when a shuttle PCR is not feasible. For optimizing PCR conditions, please refer to Section VIII (1). <LightCycler > Shuttle PCR standard protocol Stage 1: Initial denaturation sec. 20 / sec. 1 cycle Stage 2: PCR (See figure on the left) 95 5 sec. 20 / sec sec. 20 / sec. 40 cycles Stage 3: Melt Curve Analysis 95 0 sec. 20 / sec sec. 20 / sec sec. 0.1 / sec. URL: 9

10 <LightCycler 480 System> Shuttle PCR standard protocol Denature sec. (Ramp Rate 4.4 /sec.) 1 cycle PCR Analysis Mode: Quantification 95 5 sec. (Ramp Rate 4.4 /sec.) sec. (Ramp Rate 2.2 /sec., Acquisition Mode : Single) 40 cycles Melting Analysis Mode: Melting Curves 95 5 sec. (Ramp Rate 4.4 /sec.) 60 1 min. (Ramp Rate 2.2 /sec.) 95 (Ramp Rate 0.11 /sec., Acquisition Mode : Continuous, Acquisitions : 5 per ) 1 cycle Cooling sec. (Ramp Rate 2.2 /sec.) 1 cycle 3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if an assay will be performed. Refer to the instrument manual for analytical methods. 10 URL:

11 <For Smart Cycler II System> 1. Prepare the PCR mixture shown below. (Per reaction) Reagent Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 12.5 µl 1 PCR Forward Primer (10 µm) 0.5 µl 0.2 µm *1 PCR Reverse Primer (10 µm) 0.5 µl 0.2 µm *1 Template (<100 ng) *2 2 µl dh2o (sterile distilled water) 9.5 µl Total 25 µl *1: A final primer concentration of 0.2 µm is most likely to yield a good result. Nevertheless, if there is an issue with reactivity, try to find an optimal concentration between 0.1 and 1.0 µm. *2: The optimal quantity depends on the number of target copies in the template solution. Make serial dilutions to determine the appropriate amount. It is preferable to use no more than 100 ng of DNA template. Furthermore, if cdna (RT reaction mixture) is added as template, the template volume should be no more than 10% of the PCR mixture. 2. Briefly centrifuge reaction tubes with Smart Cycler centrifuge and then set them in Smart Cycler to initiate the reaction. The recommended protocol for PCR is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. Perform a 3-step PCR when using a primer with low T m value or when a shuttle PCR is not feasible. For optimizing PCR conditions, please refer to Section VIII (1). Shuttle PCR standard protocol: Stage 1: Initial denaturation Hold sec. Stage 2: PCR Number of cycles: sec sec. Stage 3: Melt Curve 3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if an assay will be performed. For analytical methods, refer to the manual for the Smart Cycler System. URL: 11

12 <For CFX96 Real-Time PCR Detection System> * Please follow the procedures provided in the manual for the CFX96 Real-Time PCR Detection System (Bio-Rad). 1. Prepare the PCR mixture shown below. (Per reaction) Reagent Volume Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 12.5 µl 1 PCR Forward Primer (10 µm) 0.5 µl 0.2 µm *1 PCR Reverse Primer (10 µm) 0.5 µl 0.2 µm *1 Template (<100 ng) *2 2 µl dh2o (sterile distilled water) 9.5 µl Total 25 µl *1: A final primer concentration of 0.2 µm is most likely to yield a good result. Nevertheless, if there is an issue with reactivity, try to find an optimal concentration between 0.1 and 1.0 µm. *2: The optimal quantity depends on the number of target copies in the template solution. Make serial dilutions to determine the appropriate amount. It is preferable to use no more than 100 ng of DNA template. Furthermore, if cdna (RT reaction mixture) is added as template, the template volume should be no more than 10% of the PCR mixture. 2. Start the reaction. The recommended protocol for PCR is the shuttle PCR standard protocol described below. Try this protocol first and optimize PCR conditions as necessary. Perform a 3-step PCR when using a primer with low T m value or when a shuttle PCR is not feasible. For optimizing PCR conditions, please refer to Section VIII (1). Shuttle PCR standard protocol Sample volume: 25 µl Step 1: sec. Step 2: PCR (See figure on the left) GOTO: 39(40 cycles) 95 5 sec sec. Step 3: Melt Curve 3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if an assay will be performed. For analytical methods, refer to the manual for CFX96 Real-Time PCR Detection System. 12 URL:

13 VIII. Appendix (1) Optimization If the recommended conditions (shuttle PCR standard protocol) provided insufficient reactivity, follow the procedures below to optimize primer concentration and PCR conditions. In addition, other real-time PCR reagent in the Perfect Real Time series (Cat. # RR820A/B, RR091A/B) may greatly improve the reactivity. PCR conditions should be selected with consideration of both reaction specificity and amplification efficiency. A PCR system well balanced between these two aspects allows accurate assays over a wide range of concentrations. System with a high reaction specificity Non-specific amplifications such as primer-dimer formation are not observed in the no template control reactions. Non-specific amplification products (those other than the target product) are not generated. System with a high amplification efficiency Amplification product is detected at earlier cycles (lower C t value). PCR amplification efficiency is high (near the theoretical maximum of 100%). [Evaluation of primer concentration] The relationships between primer concentration and reaction specificity or amplification efficiency are as follows. Reducing the primer concentration raises reaction specificities. Increasing the primer concentration, on the other hand, raises amplification efficiencies. (Primer concentration) Low (0.1 µm) High (1.0 µm) Reaction specificity High Low Amplification efficiency Low High [Evaluation of PCR conditions] To raise reaction specificity Raising the annealing temperature may improve reaction specificity. Optimize the annealing temperature while balancing specificity with amplification efficiency. [Shuttle PCR] Standard protocol Raise the annealing temperature 95 C 60 C 5 sec. 30 sec. 95 C up to 64 C 5 sec. 30 sec. To raise amplification efficiency Prolonging the elongation time or switching to a 3-step PCR may improve amplification efficiencies. Perform optimization using the steps below. [Shuttle PCR] Standard protocol 95 C 5 sec. 60 C 30 sec. Prolong the elongation time 95 C 60 C 5 sec. >1 min. 95 C 55 C 72 C [3-step PCR] 5 sec. 30 sec. 30 sec. Prolong the elongation time 95 C 55 C 72 C 5 sec. 30 sec. >1 min. URL: 13

14 Initial denaturation Generally, 95ºC for 30 sec is sufficient for initial denaturation procedures. This condition offers good reactivity in most cases, even with difficult to denature templates such as circular plasmids and genomic DNA. This procedure may be extended to 1 to 2 minutes at 95ºC depending on the template. Prolonging this procedure for too long may inactivate the enzyme. Therefore, do not heat for more than 2 minutes. [Relationship between reagent and reactivity] TAKARA BIO supplies three different real-time PCR kits for SYBR Green assay. Their respective relationships with reaction specificity and amplification efficiency are as follows. SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat. # RR420A/B) provides a high amplification efficiency and is suitable for high-speed reactions. SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. # RR820A/B) and SYBR Premix DimerEraser (Perfect Real Time) (Cat. # RR091A/B, not available in all geographic locations) are effective in raising the reaction specificity. (Reagent) SYBR Premix Ex Taq SYBR Premix Ex Taq II SYBR Premix DimerEraser (Cat. # RR420A/B/L) (Cat. # RR820A/B/L) (Cat. # RR091A/B) Reaction Specificity: Lower High Amplification Efficiency: High Lower (2) Primer design Designing a primer set with good reactivity is critical to efficient real-time PCR. Please follow the guidelines below to design a primer set that gives a high amplification efficiency and avoids non-specific reactions. 14 URL:

15 Amplification product Amplification size Primer Length The optimum size is bp. (Amplification is possible up to 300 bp in length.) mer GC content 40-60% (preferably 45-55%) T m Make sure the forward primer and the reverse primer do not differ greatly in T m values. Use software specifically designed to determine T m values. OLIGO *1 : 63-68ºC Primer3 *2 : 60-65ºC Sequence Make sure that there are no sequence biases overall. Avoid having GC-rich or AT-rich regions in the sequence (particularly at the 3 end). Avoid having consecutive T/C pairings (polypyrimidine). Avoid having consecutive A/G pairings (polypurine). 3 end sequence Avoid having GC-rich or AT-rich regions at the 3 end. It is preferable to have a G or C as the terminal base at the 3 end. It is better to avoid a primer design with T as the terminal base at the 3 end. Complementation Avoid having any complementary sequences of 3 bases or more within a primer and between primers. Avoid having any complementary sequences of 2 bases or more at primer 3' ends. Specificity Verify primer specificity by a BLAST search *3. *1: OLIGO Primer Analysis Software(Molecular Biology Insights) *2: Primer3 ( *3: (3) When performing real-time RT-PCR To perform reverse transcription reactions for real-time RT-PCR, we recommend the following products. PrimeScript RT reagent Kit (Perfect Real Time) (Cat. #RR037A/B)* PrimeScript RT Master Mix (Perfect Real Time) (Cat. #RR036A/B)* PrimeScript RT reagent Kit with gdna Eraser (Perfect Real Time) (Cat. #RR047A/B)* Any of these products in combination with this kit can yield highly reliable results. *: Not available in all geographic locations. Check for availability in your region. 1. Prepare the PCR mixture shown below. (When using Thermal Cycler Dice Real Time System) Prepare the following reaction mix at a volume sufficient for the required number of tubes plus a few extra and dispense aliquots of 22.5 to 24 µl. (Per reaction) Reagent Amount Final conc. SYBR Premix Ex Taq (2 ) (Tli RNaseH Plus), Bulk 12.5 µl 1 PCR Forward Primer(10 µm) 0.5 µl 0.2 µm PCR Reverse Primer(10 µm) 0.5 µl 0.2 µm dh2o (sterile distilled water) x µl Total 22.5 ~ 24 µl URL: 15

16 2. Add 1 to 2.5 µl of the reverse transcription reaction mixture to each of the tubes containing the reaction mixture (25 µl in total). Note: The volume of the reverse transcription reaction added to the PCR reaction should be no more than 10% of the reaction mixture. Experimental example Human ATP5F1 mrna was detected by a real-time RT-PCR assay with cdna (equivalent to 1 pg to 100 ng of total RNA) as the template and dh2o as a negative control. R 2 : Eff : 102.8% 16 URL:

17 IX. Related Products SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat. # RR420A/B) SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. # RR820A/B) SYBR Premix Ex Taq II (Tli RNaseH Plus), Bulk (Cat. # RR820L) SYBR Premix DimerEraser (Perfect Real Time) (Cat. # RR091A/B)* SYBR Premix Ex Taq GC (Perfect Real Time) (Cat. # RR071A/B)* PrimeScript RT reagent Kit (Perfect Real Time) (Cat. # RR037A/B)* PrimeScript RT Master Mix (Perfect Real Time) (Cat. # RR036A/B)* PrimeScript RT reagent Kit with gdna Eraser (Perfect Real Time) (Cat. # RR047A/B)* One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Cat. # RR066A/B)* One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (Cat. # RR086A/B)* One Step SYBR PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (Cat. # RR096A/B)* Thermal Cycler Dice Real Time System (Cat. # TP850/TP870 etc.)* Smart Cycler II System (Cat. # SC200N/SC210N) *: Not available in all geographic locations. Check for availability in your region. URL: 17

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19 NOTICE TO PURCHASER: LIMITED LICENSE [P5] PCR Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. 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. Diagnostic uses under Roche patents require a separate license from Roche. 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. [L11] SYBR Green I This product is covered by the claims of U.S. Patent No. 5,436,134 and 5,658,751 and their foreign counterpart patent claims. Takara PCR products containing SYBR Green I are sold under license from Molecular Probes Inc. only for the usage in Real-time PCR for internal research purpose. These products are not to be used for the purpose such as; providing medical, diagnostic, or any other testing, analysis or screening services or providing clinical information or clinical analysis in return for compensations. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [L46] SYBR /Melting Curve Analysis The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. [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 RNase H 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 SYBR is a registered trademark of Molecular Probes Inc. Dice is a trademark of TAKARA BIO INC. SmartCycler is a registered trademark of Cepheid Corporation. LightCycler is a registered trademark of Roche Diagnostics. Other brand names and product names, unless otherwise specified, are also proprietary trademarks or registered trademarks. URL: 19

20 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. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page at It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. If you require licenses for other use, please contact us by phone at URL: