M-MLV Reverse transcriptase

Description:

Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) uses Single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand. This enzyme is isolated from E coli expressing a portion of the pol gene of M-MLV on a plasmid.

Components:

M-MLV

5 × RT Buffer

Unit Definition:

Unit activity is calculated assuming a specific enzyme activity of 350,000 units per mg protein. Protein is determined by a modification of the Lowry method, using BSA as a standard.

One unit of M-MLV incorporates 1 nmol dTTP into acid-precipitable material in 10 minutes at 37℃,using poly(A) oligo(dT)12-18 as template primer.

Buffer component:

Storage Buffer:   

20mM Tris-HCl (pH7.5)

         1mM DTT

0.01%(v/v) Nonidet-P40

0.1mM Na2EDTA

0.1M NaCl

50%(v/v) glycerol

5 × RT Buffer

250mM Tris-HCl(pH8.3)

375mM KCl

15mM MgCl2

50mM DTT

 

Quality Control Assays:

This product has passed the following quality control assays: SDS-polycarylamide get analysis for purity; yield and length of cDNA product; functional absence of DNA endonuclease. Store the 5 × First Strand Buffer at -20℃. Thaw the solutions at room temperature just prior to use and refreeze immediately. The enclosed buffers were assayed with the enzyme and met quality control specifications.

Reaction volume

components

volume

5×RT Buffer

4μl

10mMdNTPs(10mM)

1μl

Oligod(T)12-18 (10μM)

1μl

mRNA/total RNA

5ng-500ng/50ng-5μg

M-MLV(50U/μl)

1μl

DEPC-H2O

to 20μl

    Reaction condition: 37℃, 60min, 95℃, 5min, 4℃, 5min.

cDNA can be stored at -20℃ or used in PCR directly.

Use 50 units of M-MLV per ug of RNA in a standard assay. However, reaction volume and amounts of enzyme and mRNA should be tailored to the second-strand method.

 

 

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