REDScript(TM) M-MLV Reverse Transcriptase, is an RNA-dependent DNA polymerase that synthesizes 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. This RT endures high temperature better than M-MLV RT. It can retain 100% activity at 50℃, and > 75% activity at 55℃.
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REDScript(TM) M-MLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary DNA (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 75% activity at even at 55℃.
Upper panel: Template: Mouse total RNA, Reverse transcription at 50℃
Lane 1-2: REDscript(TM) M-MLV 200U
Lane 3-4 :invitrogen SuperScript® III
Lane 5-6:Takara m-mlv
Lane M: Marker
Lower panel: Template: Mouse total RNA, Reverse transcription at 55℃
Lane 1-2:Takara m-mlv
Lane 3-4: REDscript(TM) M-MLV
Lane 5-6:200U invitrogen SuperScript® III
Lane M: Marker
Source: Recombination of E.coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.
Component: M-MLV （200U/μl）
5×Buffer （with DTT）
Features: Weak RNaseH activity High cDNA yield
Unit Definition: One unit of REDscript(TM) M-MLV RT catalyzes
the incorporation of 1 nmol of dTTP into acidinsoluble material in 10 minutes at 37℃ using oligo(dT)12-18-primed poly(A)n as a template.
Applications: The first-strand cDNA synthesis; RT-PCR.
Storage Buffer: 20 mM Tris-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
5) 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.
6) Centrifuge for a few seconds. Add 1μl REDscript(TM) M-MLV RT（200U/μl）Incubate at 50℃ for 50min.
7) Inactivate at 70℃ for 10min then get the cDNA.