BioTeke Corporation
SupermoIII M-MLV Reverse Transcriptase
Cat. #：PR 6811 PR6812
Storage: -20℃
Product description:
SupermoIII M-MuLV Reverse Transcriptase is base on M-MLV reverse transcriptase. M-MLV
reverse transcriptase is a kind of DNA polymerase depend on RNA, reaction needs DNA primers
and RNA template to synthesize integrated DNA. This enzyme is the production of rat leukemia
virus pol gene, which is a 71 kD single sub-unit. Compared with the AMV RTase, M-MLV RTase
has lower RNaseH activity; it can synthesize long DNA chains. The concentration is 200units/μl,
store at -20℃.
Product component
Supermo M-MLV
（200U/μl）
5×First-strand
Buffer
PR6811
PR6812
10000U
50000U
1 ml
1 ml×5
Product features
It has predomination to Reverse Transcription reaction that template on long mRNA (>13kb).
Thermal stability is very good, the best reaction temperature is 50。C.
Application：
 Synthesis of the first chain cDNA
 cDNA Library construction
 one-step RT-PCR
 primer extend
 3′ and 5′RACE
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Product origin
Recombinant E.coli contains Moloney murine leukemia virus reverse transcriptase gene from clone
of Moloney murine.
Activity unit
Unit activation defines that required enzyme quantity of mix into 1 nm dNTP into un-dissolution
acid during 10min at 37℃ and take 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.
Mix gently and water bath for 5 min in 70℃ in ice bath.
3.
Put the tube on 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, mix gently and then incubate in 37℃water bath for 5 min; 5 min in
25℃water bath for random primer.
4.
Centrifuge for 3-5s after mix gently. Add 1μl M-MLV RTase（200U/μl, the final volume is
20μl .
5.
Incubate in 60℃ water bath for 60 min (if use a random primer, incubate 10min in 25℃
firstly, and then 60℃ water bath in 60 min).
6.
Heat up for 10 min in 70℃, and then place on ice to next experiment or store in freezer.
PCR
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1.
Transfer 10% first reaction solution (2 μl) to a proper axenic PCR tube.
Note: the first reaction solution can directly use to PCR template without purification, dosage
is about 1-5μl. If more templates are used, the salt and random primers in first reaction
solution will restrain the activity of DNA polymerase. If need purify the first reaction solution,
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 recycle cDNA.
2.
Add next solution by order.
5μl 10×PCR Buffer
1μl
10mM dNTP mix
1μl
10μM Primer #1 (prepare youself)
1μl
10μM Primer #2 (prepare youself)
xμl
H20 (total reaction volume:49 μl)
1μl
Taq DNA polymerase
3.
Mix well and add 50μl mineral oil to the surface of liquid.
4.
Amplification program: according to annealing temperature and gene copy number and
technical parameters of Taq DNA polymerase, set program and specify references to
specification of DNA polymerase ; the usually cyclic number are 30-35.
5.
Check amplification products by agarose electrophoresis.
Telephone:
General (Ordering): +86-10- 62979408
Technical Service: +86-10-62979408
Fax: +86-10-62951781
Email:
Technical Service:bioteke@hotmail.com
Ordering Information: info@bioteke.com
Address:
No.22, Beiqing Road,
Haidian District, Beijing 100094,China P.R.
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BioTeke Corporation
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