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Description
M-MLV (H-) Reverse TranscriptaseProduct Specification Synonyms M MLV[H ] RTM MLV[H ] Reverse Transcriptase Expression System E. coli Molecular Weight 76 kDa (Reducing) Purity 95% by SDS PAGE Conjugation Unconjugated Tag His Tag Storage Buffer 20 mM Tris HCl100 mM NaCl1 mM DTT0. 1 mM EDTA0. 01% Nonidet P 4050% Glycerol pH 7. 5 @ 25C 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
Product Specification
| Synonyms | M-MLV[H-] RT，M-MLV[H-] Reverse Transcriptase |
| Expression System | E.coli |
| Molecular Weight | 76 kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Storage Buffer | 20 mM Tris-HCl、100 mM NaCl、1 mM DTT、0.1 mM EDTA、0.01% Nonidet® P-40、50% Glycerol pH 7.5 @ 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. |
Background
M-MLV (H-) Reverse Transcriptase is an RNA-dependent DNA polymerase with reduced RNase H activity. This 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(H-) reverse transcriptase has no 3´ → 5´ exonuclease activity. It can be used to synthesize first strand cDNA more efficient than the wild type M-MuLV。
Components
Storage Solution : 100 U/ul M-MLV(H-) Reverse Transcriptase、20 mM Tris-HCl、100 mM NaCl、1 mM DTT、0.1 mM EDTA、0.01% Nonidet® P-40、50% Glycerol pH 7.5 @ 25°C
5*Reaction Buffer: 250 mM Tris-HCl、375 mM KCl、15 mM MgCl2、50 mM DTT (pH 8.3 at 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(H-) 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
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