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:M-MLV Reverse Transcriptase(H-)

M-MLV Reverse Transcriptase(H-)


Description:The MMLV Reverse Transcriptase(short name MMLV) is manufacturered by MBS . Which is iolated from E.coli carring rat leukemia virus pol gene consists of a single peptide, molecule weight 71kd.It possesses RNA and DNA depended 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
Storeage: -20℃
Application:Synthesis of the first chain cDNA,  cDNA Library construction, one-step RT-PCR,  primer extention, 3′ and 5′RACE
Source:     Recombination of E.coli containing Moloney murine leukemia virus reverse transcriptase gene from 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 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 in 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 PCR template without purification ,the dosage is about 1-5μl. if excessively used ,the salt and Random primers in first reaction solution will restrain the activity of DNA polymerase .if purification needed ,it can follow the next :after reaction end of  cDNA synthesis (step 6) , add RNase A in reaction system , 10 min in 37℃ ,use DP1501 recover cDNA .
2. add 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 liquid.
4. Amplified reaction : according to annealing temperature or gene copy number or technical parameter of Taq DNA polymerase , setting amplified condition , specify reference to specification of DNA polymerase ,the usually cycle number is 30-35
5   Detect the product in agarose containing EB.

 

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