M-MLV Reverse transcripatase（RNaseH-）
Description:
Moloney Murine Leukemia Virus Reverse Transcriptase(M-MLV RT) is isolated from recombinant E.coli strain expressing the gene of M-MLV. The recombinant protein is obtained by column purification.
M-MLV (RNase H-) lacks the activity of RNase H for multiple point mutations. It has the same activity of DNA polymerase as wild type, but has obviously enhanced extension capacity. The enzyme is used to synthesize long first-strand cDNA and construct overall length of cDNA library at high proportion. The optimal reaction temperature is 42℃.
Components:
M-MLV（RNaseH-）
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（RNaseH-）（50U/μl） |
1μl |
|
DEPC-H2O |
to 20μl |
Reaction condition: 42℃, 60min, 95℃, 5min, 4℃, 5min.
cDNA can be stored at -20℃ or used in PCR directly.