Meridian’s 55C MMLV-RT is a highly robust, thermostable reverse transcriptase, ideal for cDNA synthesis from RNA with a high secondary structure such as viral RNA genomes.
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Traditional Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) is not thermostable and can only maintain its enzymatic activity at relatively low temperatures (up to 50°C). However, for cDNA synthesis, a higher reaction temperature is sometimes desirable as it reduces RNA secondary structures which can inhibit reverse transcription and it minimizes nonspecific primer binding. 55C MMLV-RT is a highly thermostable reverse transcriptase with reduced RNase H activity, that allows very efficient first-strand cDNA synthesis at temperatures up to 60°C. This improves the cDNA yield from difficult RNA targets, such as viral RNA genomes, that require higher temperature to denature strong RNA secondary structure.
|Description||Developed to reduce RNase H activity and increase thermal stability, 55C MMLV-RT can be used at temperature up to 60°C, enabling melting of areas of secondary structure in RNA, improving cDNA yield and sensitivity from difficult RNA targets such viral genomes.|
|Concentration||≥ 200 U/µL|
|Specific Activity||≥ 300,000 U/mg|
|Purity||> 95% (densitometric analysis of SDS-Page)|
|Appearance||Clear, colorless solution|
|Application||cDNA synthesis, RT-PCR, PCR, two-step RT-qPCR, one-step RT-qPCR|
|Mix stability||See outer label|
|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: 55C MMLV-RT
Yes, for RNA with very high secondary structure we recommend using 55C MMLV-RT, this thermostable reverse transcriptase can be used at temperatures up to 60°C which improves the cDNA yield from difficult RNA targets (such as viral RNA) that require higher temperature to denature strong RNA secondary structures.
55C MMLV-RT does contain the domain for RNase H, but it has been inactivated, so that there is no detectable RNase H activity.
If you are going to do RT-PCR or RT-qPCR, this is unnecessary, RNA:DNA duplex after cDNA synthesis are melted during the 95°C denaturing step at the start of the PCR reaction. For cloning of larger fragments, RNase H treatment can be beneficial.
The reverse transcriptase can be inactivated by adding a chelating agent such as EDTA or heating to 70°C or higher for 10 min.
55C MMLV can be used for first-strand cDNA synthesis at temperatures up to 60°C, providing increased specificity, higher yields and can generate cDNA up to 12 kb.
Yes, to initiate reverse transcription, the MMLV-RT reverse transcriptase require 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.
55C MMLV-based reverse transcriptase has an error rate in the range of about 4.8 × 10−5.
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