M-MLV Reverse Transcriptase is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. Compared to AMV RT, Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) lacks DNA endonuclease activity and has a lower RNase H activity. Features of this enzyme:
• Thermostability—optimal activity at 37°C
• Size of cDNA—M-MLV can be used to synthesize first-strand cDNA up to 7 kb
• Applications—synthesis of first-strand cDNA, primer extension, sequencing dsDNA, cDNA libraries, and RT-PCR
Purified from E. coli expressing the pol gene of M-MLV on a plasmid
Performance and quality testing
SDS-PAGE purity; endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product
One unit of M-MLV RT is the amount of enzyme required to incorporate 1 nmole of deoxyribonucleotide into acid-precipitable material in 10 min at 37°C using poly(A)oligo(dT)25 as template-primer.
Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 0.5 mM [3H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT)25, 0.1 mg/mL BSA, and enzyme in 50 µL for 10 min at 37°C.