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M-MuLV Reverse Transcriptase


Reverse transcriptase for routine cDNA synthesis reactions.
  
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Thermo Scientific M-MuLV Reverse Transcriptase (RT) is an RNA- and DNA-dependent DNA polymerase. It can use either RNA or DNA to prime DNA synthesis. The enzyme possesses a ribonuclease H activity specific to RNA in RNA-DNA hybrids (see References 1, 2).

Highlights

  • High yields of first strand cDNA up to 9 kb
  • Optimum activity at 37°C
  • Incorporates modified nucleotides (e.g., Cy3-, Cy5-, rhodamine-, aminoallyl-, fluorescein-labeled nucleotides)

Applications

  • First strand cDNA synthesis for RT-PCR and real-time RT-PCR
  • Synthesis of cDNA for cloning and expression
  • Generation of labeled cDNA probes for microarrays
  • DNA labeling (see Reference 3)
  • Analysis of RNA by primer extension (see Reference 3)

Includes

  • M-MuLV Reverse Transcriptase (20 U/μL)
  • 5X Reaction Buffer

Note

M-MuLV RT has significantly lower RNase H activity than Avian Myeloblastosis Virus (AMV) reverse transcriptase.

  
Storage Condition-20 C
HazardousNo
5X Reaction Buffer250 mM Tris-HCl (pH 8.3 at 25°C), 250 mM KCl, 20 mM MgCl2, 50 mM DTT
Definition of Activity Unit
  • One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [3H]-dTTP, 0.4 mM polyA·oligo(dT)12-18.
InactivationInactivated by heating at 70°C for 10 min.
InhibitionMetal chelators, inorganic phosphate, pyrophosphate, and polyamines.
M-MuLV Reverse Transcriptase (20 U/µL) supplied in50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100, and 50% (v/v) glycerol
Molecular Weight77.8 kDa monomer
Quality Control
  • The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
  • Functionally tested in first strand cDNA synthesis.
SourceE. coli cells with a cloned fragment of the pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase.

References

  1. I. M. Verma, Reverse transcriptase, The Enzymes (P.D. Boyer, ed, Academic Press Inc., vol. 14, 1981) pp. 87-103.

  2. G. F. Gerard, J. M. D’Alessio, Methods in Molecular Biology (Humana Press, N.J. Totowa, vol. 16, 1993) pp. 73-93.

  3. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, the third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).