M-MLV Reverse Transcriptase
Reverse Transcriptase for cDNA synthesis isolated from Moloney murine leukemia virus.
M-MLV Reverse Transcriptase is an enzyme isolated from M-MLV (Moloney murine leukemia virus). It is a kind of DNA polymerase used to synthesize complementary DNA strand from single stranded RNA, DNA, or RNA: DNA hybrids. It is commonly used to synthesize cDNA from RNA. It has less endonuclease activity and less RNase H activity than AMV RT.
– Purified from Moloney murine leukemia virus
• Unit Definition
– One unit of the enzyme incorporates 1 nmol dTTP into acid-precipitable material in 10 min at 37℃
– using poly(A); oligo dT as a template:primer
• Quality control
– Endonuclease Activity : 1 mg of Type I supercoiled plasmid DNA is incubated with 500 units of enzyme in 1x reaction buffer
for 1 hr at 37℃. The supercoiled DNA is visualized on EtBr-stained agarose gel to verify the absence of nicking or cutting.
– Nuclease Activity : 50 ng of radiolabeled DNA or RNA is incubated with 200 units of enzyme in 1x reaction buffer for 1 hr
at 37℃, resulting ing < 1% release for both DNase and RNase. • Physical purity – > 90% as judged by SDS-PAGE gel with coomassie blue staining
• General use
– Use 1 ml of M-MLV RT in a 20 ml of reaction.ming PCR.
[ M-MLV RTase Storage Buffer ]
• 20 mM Tris-HCl(pH 7.5)
• 0.1 mM DTT
• 0.01 % NP-40
• 0.1 mM EDTA
• 0.1 M NaCl
• 50 % glycerol
[ M-MLV Rtase 5x Reaction Buffer ]
• 250 mM Tris-HCl (pH 8.3)
• 15 mM MgCl2
• 0.1 mM DTT
• 375 mM KCl