Question 1

我的mRNA之中的GC含量较高。您建议采用哪种反转录酶？

Accepted Answer

建议您使用我们的ThermoScript 反转录酶，在如今市售的反转录酶之中，这种酶具有最高的热稳定性，可以处理您的较棘手模板（高GC含量或过多二级结构）。

Question 2

PCR反应要加入多少第一链cDNA反应产物？

Accepted Answer

尽管实际体积取决于用于第一链合成反应的RNA起始量和靶标基因的丰度，但我们建议使用10%的第一链cDNA反应产物进行PCR反应。

Question 3

My mRNA has a high-GC content. Which reverse transcriptase do you recommend using?

Accepted Answer

We would recommend using our ThermoScript Reverse Transcriptase, which provides the highest thermostability of any RT on the market to address your challenging template (high-GC content or extensive secondary structure).

Question 4

How much of the first-strand cDNA reaction should I load for PCR?

Accepted Answer

While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.

Question 5

How can reverse transcriptases be inactivated?

Accepted Answer

The enzymes can be inactivated by adding a chelating agent such as EDTA. Alternatively, with the exception of ThermoScript RT and Thermo-X RT, the enzymes can be heat inactivated at 70 degrees C for 10 min.

ThermoScript RT should be heated to 85 degrees C for 5 min for complete inactivation.

For Thermo-X RT, if using an oligo(dT) primer, add EDTA to the reaction at a final concentration of 5 mM. Inactivate the reaction by heating at 90 degrees C for 5 min.

Question 6

What is the highest temperature that SuperScript III , SuperScript II, MMLV, or ThermoScript can be used?

Accepted Answer

The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first strand synthesis temperature of 60-65 degrees C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo (dT)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.

ThermoScript™ Reverse Transcriptase - FAQs