Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase is a RNA-dependent DNA polymerase. This enzyme can synthesize a complementary DNA strand initiating from a primer using either single-stranded RNA or DNA template. The enzyme lacks RNAseH activity.
E.coli strain that carries a plasmid with the cloned and modified M-MuLVRT gene with deleted RNAseH coding part.
One unit is the amount of enzyme required to incorporate 1 nmol of dTTP into an acid insoluble form in 10 minutes at 37°C using poly(rA)-oligo(dt) 10-20 as template primer.
50 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.1 mM DTT, 0.1 mM NaCI, 0.1% Triton X-100, 50% glycerol
5X REACTION BUFFER
250 mM Tris-HCI pH 8.3, 15 mM MgCl 2, 400 mM KCl
Add to buffer: dNTPs (end concentration 2 mM), MnCl2 (end concentration 2-4 mM) and DTT (end concen-tration 10 mM). Incubate at 37°C.
25 mM MnCl2 100 mM DTT
UNIT ASSAY CONDITION
20 mM Tris-HCI pH 8.0, 2 mM MnCl2 , 100 mM KCl, 1 mM DTT, 0.6 mM poly rA, 0.1 mM poly(dT)10-20; 0.5 mM dTTP( 3 H) - 0.5-5 units of enzyme
GeneScript™ reverse transcriptase is tested for its ability to synthesize full length cDNA from 4kb RNA.
Set up a 20 µl reaction mixture as follows:
total RNA 2-5 µg; RT-buffer; primer; 1 mM each dNTP; optional: 1 U/µl RNasin
- incubate at 70°C for 2 min, chill to 23°C to anneal primer to RNA
- add 200 units GeneScript™ and incubate 10 min at 23°C followed by 30 min at 42°C
- optional: incubate with RNaseH
- heat the reaction at 95°C for 5 min and chill on ice
Store the RT-reaction by -20°C and use 2 µl for subsequent PCR.
The addition of 2 mM MnCl2 in the 1x RT buffer is optional.