MMLV Reverse Transcriptase (RNase H-) CAS 9068-38-6
Item No.： E0063
Store at： -20℃
Product name： MMLV Reverse Transcriptase (RNase H-)
Product concentration：200U/μl
Storage conditions: - 20 ℃, valid for two years.
Product Description: Reverse transcriptase can synthesize complementary DNA strands starting from RNA or single stranded DNA.MMLV Reverse Transcriptase (RNase H -) is a multi-point mutant of m-mlv reverse transcriptase. The point mutation removes the active center of RNase H, reduces the activity of RNase H, reduces the degradation of RNA in reverse transcription reaction, and improves the heat resistance of the enzyme. When reverse transcription at 50 ℃, it maintains 100% activity; It is easier to open the advanced structure of the template, increase the yield of the first strand of cDNA and obtain the full-length cDNA more easily.
Source: E. coli strain containing mutant Moloney mouse leukemia virus (m-mlv) reverse transcriptase gene.
Activity definition: With poly (a) as template and oligo (DT) as primer, the enzyme amount required for catalytic incorporation of 1nmol dTTP within 10 minutes at 37 ℃ is defined as one active unit (U).
Product application: Synthesis of the first strand cDNA; RT-PCR reaction.
20mm Tris HCl (ph7.5), 200mm NaCl, 0.25mm EDTA, 0.01% NP-40 (V / V), 2.5mm DTT, 50% Iodophor (V / V).
Product purity: 200u of this enzyme and 1 μg of 16S and 23S rRNA were incubated at 37 ℃ for 1h, and the electrophoretic bands had no visible changes.
Reaction buffer：[5×RT Buffer] 250mM Tris-HCl (pH 8.3)，15mM MgCl2，375 mM KCl，50mM DTT。
Recommended reaction conditions:
Synthesis of the first strand of cDNA
1) Oligo dt12-18 (1) was added to the PCR tube without RNase μ g/ μ l) Or gene specific primer 2-10pmol or random primer (50-250ng) 1 μl； Total RNA (10ng-5 μ g) Or mRNA (1-500ng) x μ l； dNTP (10mM each)1 μ l； DEPC ddOH2 (14-x) μl.
2) After holding at 70 ℃ for 10 minutes, quickly ice bath for 2-10 minutes.
3) Centrifugation for several seconds allowed the reaction solution to accumulate at the bottom of the PCR tube.
4) Add: 5 to the above reaction solution × RT Buffer 4 μl； RNasin (40U/ μl) 1 μl； Gently suck and mix.
5) Heat preservation at 50 ℃ for 2 minutes (oligo dt12-18 or gene specific primer); If it is random primer, keep it at 25 ℃ for 10 minutes.
6) Add 1 μl MMLV reverse transcriptase, after mixing, keep it at 50 ℃ for 50 minutes.
7) After holding at 70 ℃ for 15 minutes, it was cooled on ice to obtain cDNA. The product can be directly used for the synthesis of the second chain or RT-PCR amplification reaction. Please keep it at - 20 ℃.
