Directions for Use
AMV Reverse Transcriptase
Code
Description
Size
J398-200U
AMV Reverse Transcriptase
200 U
General Information
VWR Life Science AMRESCO’s AMV Reverse Transcriptase is an RNA-directed DNA
polymerase with 5´ - 3´ activity that is used primarily in first- and second-strand complementary
DNA (cDNA) synthesis. It may also be used in sequencing of RNA transcripts and the
preparation of probes for hybridization. AMV Reverse Transcriptase is purified from avian
myeloblastosis virus particles and possesses some intrinsic RNase H activity. The enzyme
activity requires the use of a primer (DNA primers are more efficient that RNA primers), a RNA
or single-stranded DNA template, and either Mg2+ or Mn2+.
Unit Definition: One unit is the amount of enzyme required to catalyze the transfer of 1 nmol of
deoxynucleotide into acid-insoluble material in 10 minutes at 37°C.
Reaction conditions: 50 mM Tris-HCl pH 8.3 ( at 25°C), 8.75 mM MgCl2, 40 mM KCl, 10 mM
DTT, 0.1 mg/mL acetylated BSA, 1 mM radiolabeled dTTP, 0.25 mM poly(A):oligo(dT)
Storage/Stability
Store frozen (0 to -20°C). Avoid multiple freeze-thaw cycles.
Product Use Limitations
For research use only. Not for therapeutic or diagnostic use.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
Materials Supplied
J362-200U
J395-400UL
AMV Reverse Transcriptase
 Concentration: 10 U/µL
 Source: avian myeloblastosis virus particles
 Enzyme Storage Buffer: 200 mM potassium phosphate pH 7.2 (at 4°C), 0.2%
Triton® X-100, 2 mM DTT, 50% glycerol
AMV Reverse Transcriptase Buffer 5X
 Composition: 250 mM Tris-HCl pH 8.3 (at 25°C), 250 mM KCl, 50 mM
MgCl2, 50 mM DTT, 2.5 mM spermidine
Required Materials Not Supplied
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10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP in water)
RNase inhibitor, recombinant
Sodium pyrophosphate, 40 mM
Oligo(dT) or random hexamer primers
Nuclease-free water
50 mM EDTA
[-32P]dCTP (> 400 Ci/mmol, 10 mCi/mL)
2 µg RNA
Protocol/Procedure
Notes: AMV Reverse Transcriptase requires the presence of either Mg2+ or Mn2+ ions to
function. The enzyme possesses an intrinsic RNase H activity. This product is intended for use
in standard first-strand cDNA synthesis reactions or sequencing of RNA transcripts.
First-Strand Synthesis of mRNA to cDNA
1. In a sterile, nuclease-free microcentrifuge tube, combine 2 µg RNA and 0.5 µg primer/µg
RNA in a total volume of ≤ 11 µL in water. Do not alter the ratio of primer to template
RNA.
2. Heat to 70°C for 5 minutes.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
3. Chill tube on ice for 5 minutes, then centrifuge briefly to collect the solution at the bottom
of the tube.
4. Add the following components to the annealed primer / template in the order shown
below and gently flick the tube to mix.
Component
Volume
AMV Reverse Transcriptase Buffer, 5X
5 µL
10 mM dNTP Mix
2.5 µL
RNase Inhibitor
40 units
Sodium Pyrophosphate, 40 mM (pre-warmed to 42°C)
2.5 µL
AMV Reverse Transcriptase
30 units
Nuclease Free Water
As needed
Total Volume
25 µL
5. Optional Labeled (Tracer) Reaction: Transfer 5 µL of the reaction mixture to another
tube containing 2 – 5 µCi [-32P] dCTP. Do not add label to the remaining 20 µL
reaction.
6. Incubate for 60 minutes at 42°C for oligo (dT) primers or at 37°C for random hexamer
primers.
7. Place both the labeled (tracer) and unlabeled reactions on ice and add 95 µL of 50 mM
EDTA to only the labeled (tracer) reaction, which should then total 100 µL. The labeled
reaction may be used for an incorporation assay and gel analysis.
8. Perform second-strand synthesis using the unlabeled first-strand reaction. No phenol
extraction or ethanol precipitation is necessary.
Incorporation Assay
Notes: Trichloroacetic acid (TCA) assays of isotope incorporation are prone to variation. A
yield of at least 12% from first-strand conversion should be sufficient for constructing a cDNA
library.
Required Materials Not Supplied
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1 mg/mL carrier DNA
TCA, 5% (w/v), ice-cold
Glass fiber filters (Whatman® GF-A of GF-C)
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
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Acetone or ethanol, 100%
1. Spot 3 µL of diluted first-strand tracer reaction (First-Strand Synthesis, Step 7) onto a
glass fiber filter and allow to air-dry. This sample will represent the total cpm in the
reaction.
2. Add 3 µL of diluted first-strand tracer reaction to a tube containing 100 µL of a 1 mg/mL
solution of carrier DNA and mix.
3. Add 0.5 mL of 5% TCA (ice-cold) and vortex. Keep the tube on ice for 5 – 30 minutes.
4. Filter the sample through a glass fiber filter, washing 3 times with 5 mL of 5% TCA (icecold).
5. Rinse the filter with 5 mL of acetone or ethanol and allow to air-dry.
6. Count both the total (Step 1) and incorporated (Step 5) samples, either by Cerenkov
radiation (without scintillation) or after adding an appropriate scintillation fluid.
Calculation of First-Strand Yield
Incorporated cpm
Total cpm
x 100% = % first-strand incorporation
4 (nmol dNTP/µL) x reaction volume µL x % first-strand incorporation
100
nmol dNTP incorporated x 330 ng/nmol = ng cDNA synthesized
ng cDNA synthesized
ng mRNA in reaction
= nmol dNTP incorporated
x 100% = % mRNA converted to cDNA
Note: The minimum quality control specification for first-strand conversion is 12%.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
Frequently Asked Questions
Question
Cause
RNase contamination
High secondary structure of
mRNA template
Why is the yield of cDNA low?
Suboptimal primer
concentration used.
Inhibitory components (SDS,
EDTA, salts, etc.) in synthesis
reaction.
What is the average size
range of cDNA that is
synthesized?
Typically 350-6,000 bases.
Is AMV Reverse
Transcriptase native or
recombinant?
Native
Solution
Minimize RNase contamination by using
clean, aerosol resistant tips, RNase
inhibitor, etc.
Use intact mRNA template. Check for
mRNA integrity by electrophoresis
Heat denature mRNA to 70°C for 5-10
minutes, followed by immediate chilling
on ice.
Use 0.5 µg primer per each µg mRNA
for Oligo (dT) or Random Hexameric
Primers and mRNA samples. For
control RNA, use a 1:1 ratio of primer to
RNA.
Check for inhibition of the first strand
reaction by adding a small amount of
mRNA to the positive control RNA.
Perform gel analysis of cDNA using the
tracer reaction on a 1.4% alkaline
agarose gel. Generally, 5-10 µL of the
control tracer reaction on a fixed and
dried gel results in a dark, sharp signal in
a 3-4 hour exposure with intensifying
screens.
This enzyme is purified from Avian
myeloblastosis virus.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
For Technical Support
Toll Free: 1-800-610-2789 (USA & Canada)
Fax: (440) 349-0235
Email: [email protected]
AMRESCO, LLC
A VWR Company
Corporate Headquarters
6681 Cochran Road
Solon, Ohio USA 44139-4300
Tel: 440/349-1199
Fax: 440/349-1182
www.amresco-inc.com
AMV Reverse Transcriptase
ZY0684
Rev. 0 2/2015
© Copyright 2010 by AMRESCO, LLC
All Rights Reserved.
®
AMRESCO is a registered trademark of AMRESCO, LLC
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139