M-MLV reverse transcriptase H-

MMLV Reverse Transcriptase, from Murine Leukemia virus, is an RNA-dependent DNA polymerase. It synthesizes the cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. MMLV RT also can extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some RNA templates without an additional DNA polymerase. M-MLV RT (H-) can synthesize 9.5kb products, the largest RNA component in the reaction. However, M-MLV RT synthesized more Full-length cDNA regardless of size.

long time at -20°C

One unit of activity is the amount of enzyme required to incorporate 1 nmole of dNTP into an acid-insoluble form in 10 minutes at 37°C using polyA-oligo (dT) as template and primer.

TrisHCl pH 8.3, KCl, MgCl₂, DTT

50mM Tris-HCl pH8.3, 100mM NaCl, 1mM EDTA, 0.1mM DTT, 0.1% Triton X-100, 50% glycerol

200 units of enzyme are used to produce cDNA from 1µg of a 1.2kb control RNA. The minimum specification is 120ng of first strand cDNA made from 1µg of RNA. The cDNA product must be >90% full length.

To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 500 units of M-MLV Reverse Transcriptase in 1X Reaction Buffer for one hour at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting (analysis on 0.4µg of DNA).

The purity is >90% as judged by SDS-polyacrylamide gels with Coomassie® blue staining.