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
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
