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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The MMLV Reverse Transcriptase(short name MMLV) is manufactured by MBS . Which is isolated from E.coli carrying rat leukemia virus pol gene consists of a single peptide, molecule weight 71kd. It possesses RNA and DNA depended on polymerase activity and weak RNase H activity. 

Concentration: 200U/μl

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

Package: Bulk

Features

Weak RNaseH activity; High cDNA yield

Storage: -20℃

Application: Synthesis of the first chain cDNA, cDNA Library construction, one-step RT-PCR, primer extension, 3′ and 5′RACE

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

Unit definition: One unit is defined as the required enzyme incorporate 1 nm dNTP into a polynucleotide fraction in 10 min at 37℃, taking polyA﹒poly(dT)12-18 as template-primer.

Procedure

1. add the next reaction mixture to the ice bath tube :

1) template RNA

total RNA 0.1-5μg

or total poly(A)+mRNA 0.1-0.5μg

or unique RNA 0.01pg-0.5μg

2) primer

Oligo(dT)18 (0.5μg/μl) 1μl

Or stochastic primer（0.2μg/μl） 1μl

Or sequence especially primer 20pmol

3) RNase-free ddh2o: constant volume to 11μl

2. Gently mix and water bath for 5 min at 70℃ and chill on ice.

3. Put the tube into ice and add the next composition:

5×Reaction Buffer 4μl

RNase Inhibitor (40U/μl) 0.5μl

dNTP Mix(10mmol/L) 2μl

add water to 19ul, gently mix and then water bath for 5 min in 37℃; or for 5 min in 25℃ for random primer 

4. Spin down for a few seconds. Add 1μl M-MLV RT（200U/μl）

5. Incubate at 42℃ for 60min(if use a random primer, first incubate for 10min in 25℃

6. Inactivate at 70℃ for 10min.

PCR Reaction

1. Transfer 10% volume of first reaction solution (2 μl ) to a proper PCR tube.

note: the first reaction solution can be directly used as a PCR template without purification, the dosage is about 1-5μl. if excessively used, the salt and Random primers in the first reaction solution will restrain the activity of DNA polymerase. if purification is needed, it can follow the next: after reaction end of cDNA synthesis (step 6), add RNase A in the reaction system, 10 min in 37℃, use DP1501 recover cDNA.

2. add the next solution by order.

5μl 10X PCR Buffer

1μl 10mM dNTP mix

1μl 10μM Primer #1 (customer supplied)

1μl 10μM Primer #2 (p customer supplied)

 xμl H20 (total reaction volume:49μl)

1μl Taq DNA polymerase

3. Mix thoroughly and add 50μl mineral oil to the surface of the liquid.

4. Amplified reaction: according to annealing temperature or gene copy number or technical parameter of Taq DNA polymerase, setting amplified condition, specify the reference to the specification of DNA polymerase, the usual cycle number is 30-35

5. Detect the product in agarose-containing EB.

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