Description

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.

---