M-MLV Reverse Transcriptase, RNase H-
CAT# PRODUCT SIZE STORAGE ¼ÒºñÀÚ°¡ ÁÖ¹®
19600M-MLV Reverse Transcriptase, RNase H Minus (200U/ul)10000U-20¡ÆC-·Î±×ÀÎ
19601M-MLV Reverse Transcriptase, RNase H Minus (200U/ul)20000U-20¡ÆC-·Î±×ÀÎ

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.
 

Applications

First strand cDNA synthesis
Primer extension
RT-PCR

PROTOCOL


1. 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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