M-MuLV Reverse Transcriptase, RNase H^-

High sensitivity enzyme for cDNA synthesis  

Description

Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase RNase H^- is an RNA-directed DNA polymerase used for synthesizing a complementary DNA strand. Copying is initiated from a primer using either single-stranded RNA or single-stranded DNA template.

M-MuLV reverse transcriptase RNase H^- is purified from a strain of E.coli carrying the Reverse Transcriptase gene from M-MuLV, modified by site-directed mutagenesis to eliminate the RNase H activity. M-MuLV RNase H^- RT has improved efficiency for full length cDNA synthesis.

Applications

• cDNA synthesis from single-stranded RNA or DNA.
• Multiple transcripts can be analyzed from a single RT-reaction
• Stand alone enzymes are useful when the same cDNA source is used for analysing several different genes. 

Advantages

• High sensitivity
• Improved efficiency for full length cDNA synthesis
• Purified from a recombinant source

Components

M-MuLV RT RNase H^- is supplied with 10 x M-MuLV Buffer

Reaction buffer concentrate
1 x M-MuLV Reverse Transcriptase buffer: 50 mM Tris-HCl (pH 8.3), 30 mM KCl, 8 mM MgCl2, 10 mM dithiothreitol. Supplement with dNTPs (not included).

Storage buffer
50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 % NP40, and 50 % glycerol (v/v).

Storage stability
Stable for at least one year. Recommended storage temperature –20°C.

Quality control

Unit definition
One unit is defined as the amount of enzyme required to incorporate 1 nmole of dTTP into an acid precipitable form in 10 minutes at 37 °C using poly(A)-oligo(dT) as template primer.

Activity assay conditions
50 mM Tris-HCl (pH 8.3), 8 mM MgCl2, 10 mM dithiothreitol, 0.5 mM 3H-TTP, 0.4 mM poly (rA)•oligo (dT) 12-18.

M-MuLV Reverse Transcriptase, RNase H- is tested for its ability to synthesize full length cDNAs (9kb) from crude or purified RNA templates. Purified free of detectable levels of RNase, endonuclease and exonuclease activities.