M-MuLV Reverse Transcriptase

M-MuLV-Reverse Transcriptase: The enzyme that synthesizes a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single stranded DNA as a template

Description
M-MuLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (M-MuLV RT) is an RNA-dependent DNA polymerase that synthesizes the cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. M-MuLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase.  

Applications:
– RT PCR
– Synthesis of cDNA
– mRNA 5’-end Mapping by Primer Extension Analysis
– End-labeling of DNA
– Dideoxynucleotide Sequencing

Concentration: 200 u/µl

Storage Buffer:
200 mM potassium phosphate (pH 7.2), 0.2% Triton X-100, 2 mM DTT and 50% glycerol

Reaction Buffer 5X:
250 mM Tris-HC1(pH8.3), 375 mM KCl, 15 mM MgCl₂  and 50 mM DTT

Unit definition:
One unit of the enzyme incorporates 1 nmol dTTP into acid-precipitable material in 10 minutes at 37°C, using poly(A) oligo dT as a template primer.

Transportation: on blue ice

Storage: at -20°C for 24 months

Quality control:
Endonuclease Activity:  1 µg of Type 1 supercoiled plasmid DNA is incubated with 500 units of enzyme in 1X reaction buffer for one hour at 37°C. The supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify absence of nicking or cutting.

Nuclease Activity:  50 ng of radio labelled DNA or RNA is incubated with 200 units of enzyme in 1X reaction buffer for one hour at 37°C, resulting in <1% release for both DNase and RNase.

Purity:  >90% as judged by SDS-polyacrylamide gels with blue staining. MMLV RT is free of detectable RNase, and DNase (exo- and endonuclease) activities.

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