RNase-Tolerant MMLV-RT is a reverse transcriptase/RNase inhibitor mix optimized for one-step RT-qPCR. RNase-Tolerant MMLV-RT is used for the detection of RNA at very low levels, it can also be used with a broader temperature range, making it ideal for applications such as blood bank or transplant viral testing.
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A Moloney Murine Leukemia Virus (MMLV) Reverse Transcriptase (RT), which exhibits high sensitivity and efficiency, and RNase Inhibitor (Ribonuclease Inhibitor) which inhibits a broad spectrum of eukaryotic RNases, including RNases A, B and C. RNase-Tolerant MMLV-RT shows no inhibition of polymerase or reverse transcriptase activity, so can be used in cDNA synthesis or one-step RT-qPCR reactions. RNase-Tolerant MMLV-RT is useful in any applications where the presence of RNases is a potential problem.
|Description||RNase-Tolerant MMLV-RT (Moloney Murine Leukemia Virus Reverse Transcriptase) for the detection of RNA at very low levels, it can also be used with a broader temperature range, making it ideal for applications such as blood bank or transplant viral testing.|
|Appearance||Clear, colorless solution|
|Application||cDNA synthesis, RT-PCR, PCR, two-step RT-qPCR, one-step RT-qPCR|
|Mix stability||See outer label|
|Consistency||± 0.5 Ct variance between test and reference sample|
|DNA contamination||None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets.|
|DNase/RNase Contamination||No detectable degradation|
FAQs: RNase-Tolerant MMLV-RT
For RNA with very high secondary structure, we recommend using 55C MMLV-RT (MDX117), this is a glycerol-free reverse transcriptase can be used at temperatures up to 60°C which improves the cDNA yield from difficult RNA targets that require higher temperature to denature strong secondary structures. You can add RNase Inhibitor (MDX056) to increase the RNase tolerance.
If you are going to make cDNA, you will require a buffer that contains 0.2 mM each dATP, dCTP, dGTP & dTTP, 10 mM DTT (dithiothreitol), 25 mM KCl, 3.5 mM MgCl2 and 50 mM Tris–HCl (7.5). However, if you want to do RT-qPCR, we recommend 1-Step RT-qPCR Buffer (MDX034).
The reverse transcriptase can be inactivated by adding a chelating agent such as EDTA or heating to 70°C or higher for 10 min.
Yes, to initiate cDNA synthesis, the RNase-Tolerant MMLV-RT reverse transcriptase requires a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand. For general cDNA synthesis, random hexamer primers and oligo(dT) primers can be used either separately or mixed. For RT-PCR and RT-qPCR gene-specific primers should be used.
Unlike eukaryotic replicative polymerases, reverse transcriptase lacks exonuclease activity and so is error prone. MMLV-based reverse transcriptase has been reported to have an error rate in the range of 1.1 × 10−4 to 4.8 × 10−4.
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