M-MLV Reverse Transcriptase
(E.C.2.7.7.49)
Catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids
Source:
E. coli strain containing an overproducing clone of M-MLV Reverse Transcriptase.
Description:
M-MLV Reverse Transcriptase catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids(1). Full-length copies of large mRNAs, >10 kb, may be synthesized. M-MLV Reverse Transcriptase has a much lower RNase H activity than AMV Reverse Transcriptase resulting in high yields of full length cDNA(2). This makes M-MLV Reverse Transcriptase very useful in cDNA synthesis and RT-PCR.
For added convenience and optimal RT-PCR try USB’s RT-PCR Kits (PN 78350 and 78355) and RT-PCR Master Mixes (PN 71185 and 78370).
Properties:
Molecular Weight: 71 kDa (monomeric).
Inhibitors: Polyamines, phosphate, pyrophosphates, and lithium chloride(3,4).
Inactivation: 75°C for 10 min or by adding 2 μl of 0.5M EDTA for a 50 μl reaction.
Purity:
Greater than 90% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, exonucleases and ribonucleases.
Storage Buffer:
20mM Tris-HCl (pH 7.5), 0.1M NaCl, 0.1mM EDTA, 1mM DTT, 0.01% Igepal CA-630, 50% glycerol.
Assay Conditions:
The reaction mixture (25 μl) contains 50mM Tris-HCI (pH 8.3), 40mM KCl, 6mM MgCl2, 1mM DTT, 400μM poly (rA)-oligo(dT)12 - 18, 500μM radiolabeled TTP and 0.1 - 0.5 units enzyme. Incubation is at 37°C for 10 min.
Unit Definition:
One unit is that amount of enzyme required to incorporate 1 nmol of deoxynucleotide into DE-81 filter-binding material in 10 min at 37°C using poly (rA)-oligo(dT)12 - 18 as template-primer.
Concentration:
200 units/μl
Tested User Friendly™ Functional Test:
RT-PCR amplification of targets from total RNA.
Functionally Tested 5X M-MLV Reaction Buffer (1 ml included, PN 71505):
250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl2, 50mM DTT.
References:
- ROTH, M. J., TANESE, N. AND GOFF, S. P. (1985) J. Biol. Chem. 260, 9326-9335.
- SAMBROOK, J. AND RUSSELL, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor, New York, 8.48.
- GERARD, G. F. AND D'ALESSIO, J. M. (1993) Methods In Molecular Biology 16, Humana Press, NJ, 73-93.
- SAMBROOK, J. AND RUSSELL, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor, New York, A8.16.
Applications:
- RT-PCR
- Synthesis of first strand cDNA for PCR, cloning, and hybridization probes
- Filling-in and labeling the 3' termini of DNA with 5' protruding ends
- Amplification of RNA
- Primer extension assays
Storage:
Shipped on dry ice. Store at -20°C.
MSDS / Material Safety Data Sheets
M-MLV Reverse Transcriptase_MSDS
Protocol, Brief
M-MLV Reverse Transcriptase_Brief Protocol
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