AMV Reverse Transcriptase

Description:

AMV Reverse Transcriptase (AMV RT) is isolated from avian myeloblastosis virus. Molecularweight of αβ holoenzyme is 157KD. The enzyme is used to synthesize cDNA and sequencingRNA and DNA. AMV RT catalyzes the polymerization of DNA using total RNA or polyA+ RNA. The enzyme possesses an intrinsic RNase H activity for the degradation of RNA withinRNA/DNA heterozygote. Activating agent for AMV RT is Mg2+ or Mn2+ and its optimal reaction temperature is 42℃. The reaction temperature can be 37~41℃ in the case of sodiumpyrophosphate. The enzyme is used to synthesize first-strand cDNA up to 10 kb.

Components:

AMV

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

AMV(30U/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.

 

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