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Thermo-stable M-MLV Reverse Transcriptase (RNase H-)

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Product Description:

MMLV Reverse Transcriptase is able to synthesize the complementary DNA strand from a single-strand RNA template. M-MLV Reverse Transcriptase (RNase H-) is a mutant type of M-MLV Reverse Transcriptase, obtained by eliminating the active center of RNase H through multiple point mutations. The alteration decreases activity of RNase H and reduces RNA degradation in reverse transcription, which increases the yield of first-strand cDNA to get full length cDNA more easily. In addition, thermal stability of the reverse transcriptase is improved and the optimal reaction temperature is therefore 50℃.

Source and Application:

The enzyme is obtained from E.coli strain containing mutant Molony murine leukemia virus (M-MLV) reverse transcriptase gene.It could be applied in first-strand cDNA synthesis and RT-PCR

Product Detail:

Unit Definition

One activity unit (U) refers to the amount of M-MLV reverse transcriptase when catalyzes the incorporation of 1nmol of dTTP into materials in 10 minat 37℃ using oligo (dT) primed Poly (A) as a template

Storage Buffer

20 mMTris-HCl (pH7.5), 200 mMNaCl, 0.25 mM EDTA, 0.01% NP-40 (v/v), 2.5 mM DTT, 50% Glycerol (v/v)

Product Purity

No macroscopic differences on electrophoretic bands after incubatingthe mixture of 200U M-MLV Reverse Transcriptase (RNase H-) and 1μg 16S, 23S rRNA at 37℃ for 1 h

Reaction Buffer

5x RT Buffer without DTT:

250mM Tris-HCl (pH 8.3), 15mM MgCl2, 375mM KCl

Storage Condition

-20℃, three years

Recommended Reaction Conditions:

The first-strand cDNA synthesis

1) Add the following reagents to RNase free PCR tube:

Oligo dT 12-18 (1μg/μl) or gene specific Primer (2-10pmol) or random primer (50-250ng) 1μL;

Total RNA (10ng-5μg) or mRNA (1-500ng) xμl;

dNTP (10mM each) 1μl;

RNase free ddH2O (9-x)μl.

2) Incubate 10 min at 70℃and quickly chill on ice for 2-10 min.

3) Centrifuge for a few seconds

4) Add the following components into the tube and gently mix: 5×RT Buffer 4μl; 50mM DTT 4μl; RNasin (40U/μl) 1μl.

5) Incubate 2 min at 50℃ for Oligo dT 12-18 or gene specific primer or incubate 10 min at 25℃ for random primer.

6) Add 1μl M-MLV Reverse Transcriptase and mix. And then incubate 30 min at 50℃.

7) Incubate 15 min at 70℃ and chill on ice to get the cDNA. The product can be used directly in the second-strand synthesis or in the amplification of RT-PCR. Product should be kept in -20℃.

Note: if RT-PCR is conducted on shorter fragments, easy procedure can be applied: mix all reaction components together; incubate 30 min at 50 ; incubate 15 min at 70 to inactive the reverse transcriptase.

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