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).
Recombinant E. coli strain.
¡Ü 1st strand cDNA
¡Ü Primer extension.
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.
STANDARD UNIT ASSAY CONDITIONS
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
(pH8.3), 375mM KCl, 15mM MgCl2, and 100mM DTT.
0.1M KPO4 (pH7.2),
0.2% Triton X-100, 2mM DTT, and 50% glycerol. Store at -20¡É.
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,