Product Specification
| Synonyms | MMLV RT，M-MLV Reverse Transcriptase |
| Expression System | E.coli |
| Molecular Weight | 76 kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Storage Buffer | 50 mM Tris-HCl、150 mM NaCl、1 mM DTT、0.1 mM EDTA、50% Glycerol、0.1% NP-40、pH 7.6 @ 25°C |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | [1] Blain, S. W. , and S. P. Goff . "Differential Effects of Moloney Murine Leukemia Virus Reverse Transcriptase Mutations on RNase H Activity in Mg and Mn." Journal of Biological Chemistry 271.3(1996):1448-54.
[2] Narukawa, Yutaro , et al. "Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region." Protein Engineering, Design and Selection (2021).
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Background
Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is an RNA-dependent DNA polymerase. The enzyme can use RNA (when synthesizing cDNA) or single-stranded DNA as a template and initiate the synthesis of a complementary DNA strand from a primer. M-MLV reverse transcriptase has no 3´ → 5´ exonuclease activity.
Components
Storage Solution : 200 U/ul M-MLV Reverse Transcriptase、50 mM Tris-HCl、150 mM NaCl、1 mM DTT、0.1 mM EDTA、50% Glycerol、0.1% NP-40、pH 7.6 @ 25°C
10*Reaction Buffer: 500 mM Tris-HCl、750 mM KCl、30 mM MgCl2、100 mM DTT(pH 8.3 @ 25°C)
Protocol
1. Genomic DNA was removed from the extracted cell total RNA by DNase I.
2. After incubating at 37 °C for 30 minutes, add 1µl 0.5 M EDTA (to the final concentration of 5mm) and inactivate at 75°C for 10 minutes.
3. Mix RNA sample, Random Primer and 1 ul M-MLV Reverse Transcriptase in a sterile RNase-free microfuge tube.
4. Incubate the 20 μl cDNA synthesis reaction was incubated at 25℃ for 5 minutes and then at 42℃ for 1 hour.
5. Inactivate the enzyme at 65°C for 20 minutes. The cDNA product can be directly fed into the qPCR reaction or stored at -20°C.
Guidelines
Please avoid repeated freeze-thaw cycles.
Unit Definition
1 unit refers to the amount of enzyme required to catalyze the incorporation of 1 nmol of dTTP into acid-insoluble matter in 10 minutes in a 50μl total reaction system containing 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 10 mM dithiothreitol, 0.5 mM [3H]-dTTP and 0.4 mM poly(rA). oligo(dT)12-18 at 37°C, using poly(rA). oligo(dT) as the template primer.
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