Alpha Innotech
Arcturus
B-Bridge
Bio-Whitakker
Daigger
Finnzymes
ICN
Labcon
MJ Research
Sanyo-Gallenkamp
Spectronic Unicam
Spectrum
ZipGen

AMV Reverse Transcriptase

High quality enzyme for cDNA synthesis

Description

The AMV Reverse transcriptase (AMV RT), is isolated from Avian Myeloblastosis Virus as the beta holoenzyme of molecular weight 157,000 daltons, using a modification of the method described by Houts et al. (1979). AMV RTt is qualified for cDNA synthesis and also for dideoxy sequencing of DNA and RNA. AMV RT is compatible with 4 mM sodium pyrophosphate and placental RNase inhibitor in cDNA reactions. Using AMV reverse transcriptase, the reverse transcription step can be carried out at elevated temperatures up to 70°C. High temperature increases the specificity of priming and helps to increase the efficiency by reducing RNA secondary structures. AMV RT is supplied with optimized 10 x AMV Buffer.

 

Applications

bulletFirst-strand cDNA synthesis
bulletDideoxy sequencing of DNA and RNA

 

Advantages

bulletCompatible with 4 mM sodium pyrophosphate and placental RNase inhibitor in cDNA reactions

 

Components

AMV RT is supplied in storage buffer: 0.2 M KPO4 (pH 7.2), 2.0 mM DTT, 0.2 % Triton® X-100 and 50 % Glycerol (v/v), and provided with 10 x AMV Buffer concentrate which is used as 10 x or 5 x stock depending on the application.

Reaction buffer concentrate
250 mM Tris-HCl (pH 8.3 at 25 °C), 50 mM MgCl2, 500 mM KCl and 20 mM DTT. Thaw at 37 °C. Triturate with pipet tip to dissolve, if necessary.

Dilution buffer
10 mM KPO4 (pH 7.2), 2.0 mM DTT, 0.2 % Triton® X-100, 10 % Glycerol (v/v).

Storage stability
Stable for at least 1 year. Recommended storage temperature -20 °C (–70 °C for long term storage).
Note: Upon receipt, centrifuge the vial in a microfuge to ensure full recovery of the enzyme. In order to conserve optimal activity, it is strongly recommended that the enzyme be aliquoted and stored at -70 °C, although storage at -20 °C is adequate for short periods of time. Repeated freezing and thawing results in loss of enzyme activity. Due to the viscosity of the enzyme, maximum dispensing efficiency is achieved by the use of positive displacement pipets.

Quality conrol

Unit definition
One unit is defined as the amount of AMV RT required to catalyze the incorporation of one nmol of dTMP into an acid-insoluble product in 10 minutes at 37 °C using Poly(rA)•(dT)12-18 as a template primer.

Activity assay conditions
50 mM Tris-HCl (pH 8.3 at 25 °C), 6.0 mM MgCl2, 40 mM KCl, 4.0 mM DTT, 0.5 mM [3H]-TTP (10-20 cpm/pmol), 0.4 mM Poly(rA)•(dT)12-18 and 2 - 5 units AMV RT.

Ribonuclease Assay
This enzyme is free of exogenous RNase. Fifteen units of AMV RT were incubated with 1 µg of RNA Ladder for two hours at 37 °C, in 1 x reaction buffer. No change in the electrophoretic pattern of the RNA was detected. This assay is capable of detecting 2 x 10-8 Units of RNase 1A.

Exonuclease Activity
Incubation of 15 U for 4 hours at 37 °C in 50 µl assay buffer with 1 µg sonicated [3H] DNA (2 x 105 cpm/mg) released < 2 % of radioactivity.

Endonuclease Assay
No endonuclease activity is observed after incubation of 15 U of AMV RT with 1 µg of DNA-Hind III DNA fragments in assay buffer at 37 °C for 4 hours

 


Your text here!~

 


 



Your text here!

 




   

Home  |  Table of Contents  |  Contact Us