Description
M-MLV RT, H- (Molony Murine Leukemia Virus
Reverse Transcriptase, RNase H Minus), a point mutant of M-MLV RT, is an
RNA-dependent DNA polymerase that can be used in cDNA synthesis with long RNA
templates (>6kb). It¡¯s engineered to be RNase H Minus, delivering high
yields and full-length cDNA. Also M-MLV RT, H- has full activity at 42~45¡É.
You¡¯ll get even higher cDNA yields and you can work at higher temperatures for
superior performance in all your RT experiments.
Source
Recombinant E. coli strain
Concentration
200 U/§¡
Storage buffer
20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.1
mM EDTA, 1 mM DTT, 0.01 % (v/v) Nonidet P-40, 50 % Glycerol
5X reaction buffer
250 mM Tricine-KOH (pH 8.9), 50 mM
(NHl4)l2SO4, 100 mM KCl, 50 mM DTT, 15 mM MgCl2
Storage condition
-20¡É
Features
M-MLV RT, H- is a point mutant of M-MLV RT.
This enzyme lacks RNase H activity but retains wild-type polymerase activity,
so it can make more specific products than wild-type M-MLV RT.
Quality control tests
Activity, SDS-PAGE/purity, dsDNase, RNase,
endonuclease/nickase, specific performance test
Activity unit definition
One unit incorporate 1 nmol of
deoxynucleotide into acid-precipitable material in 10 min at 37¡É using
poly(A)oligo(dT)12-18 as template-primer.
Purity definition
Single 71 kDa band was visible in SDS-PAGE.
ApplicationsFirst strand cDNA synthesis
Primer extension
RT-PCR PROTOCOL1. A typical procedure uses between 1 ng ~ 2 ug total RNA or 10 pg ~ 1 ug mRNA 2. Thaw template RNA, primers, dNTPs, RT buffer, and RNase-free water, and place them on ice 3. In a sterile RNase-free microcentrifuge tube, add primer (0.5 ug oligo(dT)12-18, 50 ~ 250 ng random primer, or 2 pmol of gene-specific primer) and 3.2 ul of 2.5 mM dNTPs to the RNA 4. Heat the tube to 70 ¡ÆC for 5 min, then cool quickly on ice 5. Add 4 ul of RT buffr, 1 ul of M-MLV RT(H-), and RNase-free water up to 20 ul 6. Mix gently. Incubate a reation using oligo(dT)12-18 or gene-specific primer at 42~45 ¡ÆC for 50 min. For a reaction using random primer at 25 ¡ÆC for the initial 10 min, then 42 ¡ÆC for the final 50 min 7. Inactivate the reaction by heating for 15 min at 70 ¡ÆC. The cDNA can now be used as a template for amplification by PCR 19600_19601.pdf (127,757kb) |
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