Description
M-MuLV Reverse Transcriptase is a DNA polymerase which uses RNA as a substrate and exhibits no measurable proofreading 3ʹ to 5ʹ exonuclease function. This enzyme can perform cDNA synthesis by extending a DNA primer annealed to an RNA template and can also copy a single-stranded DNA template. The RNase H domain can act independently of the polymerase activity. Competition between polymerase and RNase H activities can limit efficiency of cDNA synthesis. DNA primer or incomplete cDNA of forming duplex may serve as RNase H substrate. The enzyme is supplied in 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40 Alternative and 50% glycerol; pH 7.6 @ 25°C. Please inquire about REACH compliant formulations. The 10x M-MuLV RT Buffer (cat. no. B7040) contains 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl 2 and 100 mM DTT; pH 8.3 @ 25°C.
Properties Storage temperature: –25°C to –15°C Recommended reaction temperature: below 42°C Transcription length: cDNA up to 7 kb Molecular weight: 68,100 Daltons Test Specification Purity >99% Specific activity 169,000 U/mg Single-stranded exonuclease 2000 U; <5.0% released Double-stranded endonuclease 2000 U; <1.0% released Double-stranded endonuclease 2000 U; no conversion E. coli DNA contamination 2000 U; <10 copies RNase contamination 2000 U; no detectable non-specific RNase
The source of the protein is a recombinant E. coli strain carrying the Moloney-Murine Leukemia Virus Reverse Transcriptase gene. One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using poly r(A)/oligo (dT) as a substrate.
First-strand synthesis 1. Primer annealing: Combine the following in an RNase-free reaction vessel: Amount Description Final concentration 1 µL 25 mM dNTP Solution (cat. no. N2050L) 2.0 mM Variable 1–2 µg total RNA or 5–500 ng mRNA (poly-A selected) Variable 1 µL Oligo (dT) 12–18 (50 µg/mL) or Random primers (125 µg/mL) or GSP primer (2 pmol) Variable Nuclease-free water to bring the total volume to 10 µL 2. Heat reaction for 5 minutes at 65°C. Spin briefly (5 seconds) to pull down condensate and place immediately on ice. 3. Add 1 µL 10x M-MuLV RT Buffer (cat. no. B7040) and nuclease-free water to a final volume of 10 µL per reaction. 4. Incubate as follows: • If using oligo (dT) or GSP primers, 2 minutes at 42°C. • If using random primers, 2 minutes at 25°C. 5. Add 1 µL (200 units) M-MuLV Reverse Transcriptase (cat. no. P7040) and mix by gently pipetting sample. (Note: if using random primers, pre-incubate reaction at 25°C for 10 minutes). 6. Incubate at 42°C for 45–60 minutes. 7. Inactivate enzyme at 85°C for 10 minutes. 8. Store products at –20°C or proceed to next step. QUALITY CONTROL ANALYSIS Unit activity is measured using a twofold serial dilution method. Dilutions of enzyme are made in 1x M-MuLV RT Buffer and added to 50 µL reactions containing 20 µg/mL poly r(A) RNA, oligo (dT) DNA, 1x RT Buffer, 3H-dTTP and 250 µM dTTP. Reactions are incubated 10 minutes at 37°C, placed on ice and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25–A8.26). Protein concentration is determined using the Bio-Rad Protein Assay Kit II (cat. no 500-0002). E. coli 16S rDNA contamination is measured in 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The non-specific RNase assay uses the RNase Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample. Single-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.
