DESCRIPTION
MMLV (Molony Murine Leukemia Virus) Reverse Transcriptase is a DNA polymerase that synthesizes a complementary DNA strands from single-stranded RNA, DNA, or an RNA-DNA hybrid as a template. This enzyme requires a primer to intiate synthesis and Mg2+ or Mn2+ for activity. Compared to AMV Reverse Transcriptase, this enzyme has a much weaker 5' ¡æ 3' ribonuclease H activity, which allows the syntesis of longer cDNAs (>7kb).
SOURCE
Recombinant E. coli strain.
APPLICATIONS
¡Ü 1st strand cDNA synthesis.
¡Ü Primer extension.
¡Ü RT-PCR.
UNIT DEFINITION
One unit is defined as the amount of enzyme required to catalyze the incorporation of 1nmol of deoxyribonucleotide into acid-insoluble forms in 10 minutes at 37¡É, using poly(A)-oligo(dT)12-18 as the template-primer.
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STANDARD UNIT ASSAY CONDITIONS
50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM each dATP, dGTP, dCTP, and dTTP, 1-10 ¥ìCi of [¥á32P]dCTP added as a tracer, 10¥ìg/ml oligo (dT)12-18, 20¥ìg/ml mRNA, 200units M-MLV RT. The reaction volume was 20¥ìl and the incubation was 60 min at 37¡É.
CONCENTRATION: 200 units/¥ìl.
5X REACTION BUFFER 250mM Tris-HCl (pH8.3), 375mM KCl, 15mM MgCl2, and 50mM DTT.
STORAGE CONDITIONS
0.1M KPO4 (pH7.2), 0.2% Triton X-100, 2mM DTT, and 50% glycerol. Store at -20¡É.¡¡
REFERENCES
1. Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
2. Gerald, G. F. and D'Alessio, J. M. (1993) In Methods in Molecular Biology, Vol. 16, Humana Press, NJ. |