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M-MuLV Reverse Transcriptase

#EP0351 Supplied with: 5X Reaction Buffer	1000 u (20 u/µl) 1 ml
#EP0352 Supplied with: 5X Reaction Buffer	5000 u (20 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0351, #EP0352 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Suitable for synthesis of full-length cDNA as long as 9 kb.
• Incorporates modified nucleotides.

Description
The 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 (1, 2).

Source
E.coli cells with a cloned fragment of the pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase.

Molecular Weight
77.8 kDa monomer.

Applications

• Generation of first strand cDNA (3) for use in:
• PCR, see Protocol for First-strand cDNA Synthesis;
• real-time PCR;
• second strand cDNA synthesis.
• DNA labeling (3).
• Analysis of RNA by primer extension (3).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests. Functionally tested in first strand cDNA synthesis.

Concentration
20 u/µl

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), 6 mM MgCl₂, 10 mM DTT, 40 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [³H]-dTTP, 0.4 mM polyA•oligo(dT)_12-18.

Storage Buffer
The enzyme is supplied in: 50 mM Tris-HCl (pH 8.3), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

5X Reaction Buffer
250 mM Tris-HCl (pH 8.3 at 25°C), 250 mM KCl, 20 mM MgCl₂, 50 mM DTT.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate, pyrophosphate and polyamines (2).
• Inactivated by heating at 70°C for 10 min.

Note
M-MuLV Reverse Transcriptase has much lower RNase H activity than Avian Myeloblastosis Virus (AMV) reverse transcriptase.

Related Products

dNTP Mix, 10 mM each RiboLock™ RNase Inhibitor First Strand cDNA Synthesis Kit Primers Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP Taq DNA Polymerase (recombinant) Taq DNA Polymerase (native): without BSA with BSA 2X PCR Master Mix High Fidelity PCR Enzyme Mix Long PCR Enzyme Mix DNase I, RNase-free DNA Polymerase I, E.coli RNase H, E.coli T4 DNA Ligase Pyrophosphatase, Inorganic (from yeast) DNA ladders RiboRuler™ RNA ladders DEPC-treated Water

References

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

2. Gerard, G.F., D’Alessio, J.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.
3. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

Updated kovo 18, 2008 09:44