MMLV Reverse Transcriptase

#3201

 

10,000 units

#3201L

 

50,000 units

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' 3' ribonuclease H activity, which allows the syntesis of longer cDNAs (>7kb).

SOURCE
Recombinant E. coli strain.

APPLICATIONS
1st strand cDNA synthesis.
Primer extension.
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, using poly(A)-oligo(dT)12-18 as the template-primer.

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 Ci of [32P]dCTP added as a tracer, 10g/ml oligo (dT)12-18, 20g/ml mRNA, 200units M-MLV RT. The reaction volume was 20l and the incubation was 60 min at 37.

CONCENTRATION: 200 units/l.

5X REACTION BUFFER
250mM Tris-HCl (pH8.3), 375mM KCl, 15mM MgCl2, and 100mM DTT.

STORAGE CONDITIONS
0.1M KPO4 (pH7.2), 0.2% Triton X-100, 2mM DTT, and 50% glycerol. Store at -20.

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.