M-MLV Reverse Transcriptase (200 U/μL), 40,000 units - FAQs

View additional product information for M-MLV Reverse Transcriptase (200 U/μL) - FAQs (28025021, 28025013)

5 product FAQs found

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

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.

How can reverse transcriptases be inactivated?

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.

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

The optimal temperature for for MMLV is 42 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. 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 IV RT is 50 degrees C, but can be used up to 65 degrees C.

Can I use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase (Cat. No. 28025013, 28025021)? Can other reverse transcriptases, such as SuperScript reverse transcriptase, be used in the same way?

Yes, you can use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase.

We have not tested this for SuperScript reverse transcriptases, so we cannot guarantee it would also work with those products.

This article can be used as a reference for additional information.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

Can I use elevated temperatures for reverse transcription with Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase?

Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase is not well-suited for elevated temperatures. Typically, M-MLV RT operates optimally at temperatures around 37 degrees C to 42 degrees C.

To perform reverse transcription at elevated temperatures (e.g., 50 degrees C or higher), consider using thermostable reverse transcriptases such as SuperScript IV or Maxima H Minus, which are engineered to perform efficiently at higher temperatures.