- RT PCR
- Synthesis of cDNA
- mRNA 5’-end Mapping by Primer Extension Analysis
- End-labeling of DNA
- Dideoxynucleotide Sequencing
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.
Concentration: 200 u/µl
200 mM potassium phosphate (pH 7.2), 0.2% Triton X-100, 2 mM DTT and 50% glycerol
Reaction Buffer complete 10X:
500 mM Tris-HCl (pH 8.3 at 25°C); 30 mM MgCl2; 750 mM KCl; 100 mM DTT.
Dilution Buffer 1X: 10 mM KH2PO4 (pH 7.5); 0,1 mM EDTA; 200 mM NaCl; 7 mM 2-mercaptoethanol; 50% glycerol
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
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
Purity: >90% as judged by SDS-polyacrylamide gels with blue staining. MMLV RT is free of detectable RNase,
and DNase (exo- and endonuclease) activities.