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分子别名
M-MLV[H-] RT,M-MLV[H-] Reverse Transcriptase表达宿主
E.coli分子量
76 kDa (Reducing)
纯度
＞95% by SDS-PAGE活性
100 U/ul标记
Unconjugated标签
His Tag缓冲体系
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
储存条件
Store at -25 ~ -15℃ for 2 years
文献引用
[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).
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。
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)
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.
Please avoid repeated freeze-thaw cycles.
- One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT)18 as template.
生物活性
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
电泳
1μg (R: reducing condition, N: non-reducing condition).
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