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55C MMLV-RT

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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Meridian's 55C MMLV-RT

  • Thermostable reverse transcriptase activity up to 60°C

  • Ideal for cDNA synthesis from RNA with high secondary structure such as viral RNA genomes

  • Sensitive detection of low copy number RNA targets

  • Perfect for developing fast, highly reproducible RT-qPCR assays

55C MMLV-RT Graph

55C MMLV-RT – MDX117

Available in 50 µL (10,000 Units), 200 µL (40,000 Units) or 1 mL (200,000 Units) aliquots

Description

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.


Specifications

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
Sample type RNA
Presentation 1 vial
Storage -20 °C
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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