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Tech Note

Specific, consistent real-time PCR with TB Green Premix Ex Taq II

Data kindly provided by: Na Wei, Ph.D., Research Associate, Scripps Research Institute

Introduction Results Conclusions Methods

Introduction  

TB Green Premix Ex Taq II (Tli RNase H Plus) is ideal for real-time PCR experiments where specificity is critical. A high level of precision is achieved and maintained across samples through effective suppression of amplification of primer dimers and off-target products. This reagent is recommended for situations where primer design has been optimized, but increased qPCR specificity is still needed.

In this study, the mRNA levels of transcription factor E2F1 were analyzed in HEK293 cells transfected with either an experimental or control plasmid. The protocol was carried out with TB Green Premix Ex Taq II (Cat. # RR820Q) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), using a 30-second extension step.

Results  

Melt curve analyses indicated a high specificity for both E2F1 and the GAPDH control (Figure 1, bottom panel). A relative quantification of the raw data was performed using the delta delta Ct method. This analysis (Figure 2) showed no significant difference in E2F1 mRNA levels between samples, indicating the capacity of the TB Green master mix to provide reliable results with low sample-to-sample variation.

Fold Expression

Figure 1. Amplification curve (top) and melt curve (bottom) for E2F1 and GAPDH (endogenous control).

E2F1 mRNA levels of HEK293 cells.

Figure 2. Relative quantification of E2F1 mRNA levels of HEK293 cells transfected with the control plasmid and the focused gene A plasmid.

Conclusions  

The use of TB Green Premix Ex Taq II resulted in high specificity and low sample-to-sample variation in real-time PCR results. This premix provided consistent results under the conditions tested.

Methods  

HEK293 cells were transfected with a control vector or a focused gene A plasmid, and RNA was isolated using Trizol Reagent (Thermo Fisher Scientific). First-strand cDNA (localizing to the cDNA of E2F1) was generated using the RT2 First Strand Kit (SABiosciences), according to the manufacturer's instructions.

The first-strand cDNA was used as template for amplification of a 150-bp fragment (corresponding to the E2F1 cDNA) via real-time PCR with TB Green Premix Ex Taq II. GAPDH was used as an endogenous control. Reaction mixtures were prepared as described in the user manual.

After reaction assembly, a StepOnePlus Real-Time PCR System was used for real-time PCR using the recommended cycling conditions in the TB Green Premix Ex Taq II user manual.

Featured products

Cat. # Product Size Price License Quantity Details
RR820A TB Green® Premix Ex Taq™ II (Tli RNase H Plus) 200 Rxns USD $348.00

License Statement

ID Number  
M40 This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.   
M82 This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.

TB Green Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using TB Green. It is supplied at a 2X concentration premixed with TB Green at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNase H, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mRNA when using cDNA as template. This product, with a modified buffer composition, offers a higher reaction specificity than that of TB Green Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and Takara Ex Taq HS, a hot start PCR enzyme that uses an anti-Taq antibody, allows highly reproducible and reliable real time PCR analyses.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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RR820A: TB Green Premix Ex Taq II (Tli RNase H Plus)

RR820A: TB Green Premix Ex Taq II (Tli RNase H Plus)
RR820B TB Green® Premix Ex Taq™ II (Tli RNase H Plus) 400 Rxns USD $635.00

License Statement

ID Number  
M40 This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.   
M82 This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.

TB Green Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using TB Green. It is supplied at a 2X concentration premixed with TB Green at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mRNA when using cDNA as template. This product, with a modified buffer composition, offers a higher reaction specificity than that by TB Green Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and Takara Ex Taq HS, a hot start PCR enzyme that uses an anti-Taq antibody, allows highly reproducible and reliable real time PCR analyses.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents
RR820L TB Green® Premix Ex Taq™ II (Tli RNase H Plus), 1 x 5 mL 200 Rxns USD $348.00

License Statement

ID Number  
M82 This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
M40 This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.   

TB Green Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using TB Green. It is supplied at a 2X concentration premixed with TB Green at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mRNA when using cDNA as template. This product, with a modified buffer composition, offers a higher reaction specificity than that by TB Green Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and Takara Ex Taq HS, a hot start PCR enzyme that uses an anti-Taq antibody, allows highly reproducible and reliable real time PCR analyses. Cat. #RR820L and RR820W are larger package sizes [5 ml and 25 ml (5 x 5 ml), respectively] of TB Green Premix Ex Taq II (Tli RNase H Plus) (#RR820A). Each of these includes a separate vial of ROX Reference Dye.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

RR820L: TB Green Premix Ex Taq II (Tli RNase H Plus), 1 x 5 mL

RR820L: TB Green Premix Ex Taq II (Tli RNase H Plus), 1 x 5 mL
RR820W TB Green® Premix Ex Taq™ II (Tli RNase H Plus), 5 x 5 mL 1,000 Rxns USD $1299.00

License Statement

ID Number  
M82 This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
M40 This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.   

TB Green Premix Ex Taq II (Tli RNase H Plus) is a reagent specifically designed for intercalator-based real time PCR using TB Green. It is supplied at a 2X concentration premixed with TB Green at a concentration appropriate for real time monitoring, making it easy to prepare reaction mixtures. The 2X premixed reagent also contains Tli RNaseH, a heat-resistant RNase H, which minimizes PCR inhibition due to residual mRNA when using cDNA as template. This product, with a modified buffer composition, offers a higher reaction specificity than that by TB Green Premix Ex Taq (Tli RNaseH Plus) (Cat.# RR420A). With inhibition of non-specific amplifications, which interfere with quantitative determination, accurate assays over a wide range are possible. A combination of this buffer and Takara Ex Taq HS, a hot start PCR enzyme that uses an anti-Taq antibody, allows highly reproducible and reliable real time PCR analyses. Cat. #RR820W contains 5 of Cat. #RR820L. Please refer to Cat. #RR820L for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents

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