Thermostable M-MLV Reverse Transcriptase (RNase H-)
Supplied by Bulk
Template: Mouse total RNA, Reverse transcript at 50℃
Lane 1-2: MBS Thermostable M-MLV 200U
Lane 3-4 :invitrogen SuperScript® III
Lane 5-6:Takara m-mlv
Lane M: Marker
Template: Mouse total RNA, Reverse transcript at 55℃
Lane 1-2:Takara m-mlv
Lane 3-4:MBS Thermostable M-MLV
Lane 5-6:200U invitrogen SuperScript® III
Lane M: Marker
Thermostable M-MLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. M-MLV RT will also extend primers hybridized to single-stranded DNA. Second-strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase. The difference between this to the general M-MLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50℃, it can also keep more than 80% activity even at 55℃.
Recombination of E.coli containing Moloney murine leukemia virus reverse transcriptase gene from the clone of Moloney murine.
Component: M-MLV （200U/μl）5×Buffer（with DTT）
lack RNase H activity: Weak RNaseH activity High cDNA yield, can get more full-length cDNA.
thermostable: the optimum reaction temperature is 50℃, the highest is 60℃. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.
wide temperature range: can reverse transcript from 37-60C, with more than 80% of the highest activity at 42℃-55℃ customer can choose the reaction temperature freely.
Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.
One unit of MMLV RT catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37℃ using oligo(dT)12-18-primed poly(A)n as a template.
The first-strand cDNA synthesis; RT-PCR.
20 mMTris-HCl (pH7.5),200 mM NaCl, 0.25 mM EDTA,0.01% NP-40(v/v),2.5 mM DTT,50%glycerol(v/v).
5X Reaction Buffer
[5×RT Buffer] 250mM Tris-HCl (pH 8.3), 15mM MgCl2,375 mM KCl,50mM DTT.
Recommended Reaction Conditions
The first-strand cDNA synthesis
1) Add the following reagents to a RNase free PCR tube at room temperature add the MMLV RT last.
Oligo dT12-18 (1μg/μl) or random primer (50-250ng) 1μl
Total RNA (10ng-5μg) or mRNA(1-500ng) xμl
dNTP (10mM each) 1μl
DEPC ddH2O (14-x)μl
2) Gently mix and incubate 10 Min at 70℃ then chill on ice for 2-10min.
3) Centrifuge for a few seconds then Put the tube into ice and add the next composition :
5×RT Buffer 4μl
RNasin (40U/μl) 1μl
4) Gently mix and incubate at 50℃ for 2 Min（Oligo dT12-18 or sequence especially primer）or at 25℃ for 10 min for the random primer.
5) Centrifuge for a few seconds. Add 1μl M-MLV RT（200U/μl）Incubate at 50℃ for 50min.
6) Inactivate at 70℃ for 10min then get the cDNA.