DescriptionM-MLV RT (Molony Murine Leukemia Virus Reverse Transcriptase) is an RNA-dependent DNA polymerase requiring a DNA primer and an RNA template to synthesize a complementary DNA strand. M-MLV RT has a weaker intrinsic RNase H activity than AMV RT (Avian Myeloblastosis Virus Reverse Transcriptase). Purified from an E. coli strain carrying a M-MLV RT overproducing plasmid.Source
Recombinant E. coli strain
Concentration
200 U/§¡
Storage buffer
20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 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 perform superior result,
efficiency, sensitivity, and linearity over a wide range of starting RNA
amounts.
Quality control tests
Activity, SDS-PAGE/purity, ds-DNase, RNase,
endonuclease/nickase, first-strand cDNA synthesis
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.
Applications
First strand cDNA synthesis
Primer extension PROTOCOL1. Add 1 ng to 5 ug of total RNA (mRNA 1 ng to 500 ng) and add 1ul of 100 pmole/ ul of oligo(dT)12-18 or random primer in RNase-free microcentrifuge tube 2. DEPC-treated sterile water up to 11ul. 3. Heat mixture to 70 ¡ÆC for 5 min and quick chill on ice 4. Add 4 ul of 5X M-MLV buffer and 4 ul of 2.5 mM dNTP mixture (each) 5. Mix contents of the tube gently and incubate at 37 ¡ÆC for 2 min 6. Add 1 ul of M-MLV Reverse Transcriptase (200 U/ ul) 7. Mix by pipetting gently up and down (total 20 ul) 8. Incubate 60 min at 37 ¡ÆC (up to 42 ¡ÆC) 9. Inactivate the reaction by heating at 70 ¡ÆC for 15 min 19500_19501.pdf (125,775kb) |
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