The nucleic acid glue map shows the effect of M-MLV(H-) reverse transcriptase on total RNA for qPCR detection. The total RNA extracted from HEK293T cells was 2µg. In 20μl reverse transcription system (1µg Total RNA, 120uM Random Hexamer primer, 1X Reaction Buffer, 20U RNase Inhibitor, dNTP Mix (1mM each), After retrotranscription, 1μl of the retrotranscription product was obtained for qPCR amplification and electrophoresis detection of 197bp segment of β-actin cDNA. As shown in the figure, this product has comparable enzyme activity compared with Competitor N's products.
M, marker
Lane 1 Negative Control-1(negative control with no added enzyme only)
Lane 2 Negative Control-2(only negative controls without templates)
Lane 3 UA-M-MLV(H-) Reverse Transcriptase 100U
Lane 4 Competing product N 100U
M-MLV (H-) Reverse Transcriptase
M-MLV (H-) Reverse Transcriptase
Product Details
Product Details
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
Picture
Picture
Bioactivity
SDS-PAGE
1μg (R: reducing condition, N: non-reducing condition).
