M-MLV Reverse Transcriptase (H+)

The MMLV Reverse Transcriptase(short name MMLV) i is iolated from E.coli carrying rat leukemia virus pol gene consists of a single peptide, molecule weight 71kd.It possesses RNA and DNA depended polymerase activity and normal RNase H activity. Concentration: 200U/μl Component : M-MLV （200U/μl） 5×Buffer （with DTT） Package： Bulk Features: NormalRNaseH activity; an alternative to the RNase H- enzyme. 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 or another fluorescent dye.