M-MLV Reverse Transcriptase
CAT# PRODUCT SIZE STORAGE ¼ÒºñÀÚ°¡ ÁÖ¹®
19500M-MLV Reverse Transcriptase (200U/ul)10000U-20¡ÆC-·Î±×ÀÎ
19501M-MLV Reverse Transcriptase (200U/ul)20000U-20¡ÆC-·Î±×ÀÎ

Description

M-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

PROTOCOL


1. 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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