Manual

Product Number: RT03

Thermostable M-MLV Reverse Transcriptase (RNase H-)

Supplied by Bulk

Description

Thermostable M-MLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. M-MLV RT will also extend primers hybridized to single-stranded DNA. Second-strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase. The difference between this to the general M-MLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50℃, it can also keep more than 80% activity even at 55℃.

Source

Recombination of E.coli containing Moloney murine leukemia virus reverse transcriptase gene from the clone of Moloney murine.

Concentration: 200U/μl

Component: M-MLV （200U/μl）5×Buffer（with DTT）

Package： Bulk

Features

lack RNase H activity: Weak RNaseH activity High cDNA yield, can get more full-length cDNA.

thermostable: the optimum reaction temperature is 50℃, the highest is 60℃. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.

wide temperature range: can reverse transcript from 37-60C, with more than 80% of the highest activity at 42℃-55℃ customer can choose the reaction temperature freely.

Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.

Storage: -20℃

Unit Definition

One unit of MMLV RT catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37℃ using oligo(dT)12-18-primed poly(A)n as a template.

Applications

The first-strand cDNA synthesis; RT-PCR.

Storage Buffer

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

5X Reaction Buffer

[5×RT Buffer] 250mM Tris-HCl (pH 8.3), 15mM MgCl2,375 mM KCl,50mM DTT.

Recommended Reaction Conditions

The first-strand cDNA synthesis

1) Add the following reagents to a RNase free PCR tube at room temperature add the MMLV RT last.

Oligo dT12-18 (1μg/μl) or random primer (50-250ng) 1μl

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

dNTP (10mM each) 1μl

DEPC ddH2O (14-x)μl

2) Gently mix and incubate 10 Min at 70℃ then chill on ice for 2-10min.

3) Centrifuge for a few seconds then Put the tube into ice and add the next composition :

5×RT Buffer 4μl

RNasin (40U/μl) 1μl

4) Gently mix and incubate at 50℃ for 2 Min（Oligo dT12-18 or sequence especially primer）or at 25℃ for 10 min for the random primer.

5) Centrifuge for a few seconds. Add 1μl M-MLV RT（200U/μl）Incubate at 50℃ for 50min.

6) Inactivate at 70℃ for 10min then get the cDNA.

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