Certificate of Analysis

AMV Reverse Transcriptase:

Part No. Size (units)
M510A 300
Part# 9PIM510
M510F 1,000
M900A (High Conc.) 600
Revised 10/16
AMV Reverse Transcriptase 5X Reaction Buffer (M515A): The AMV Reverse Transcriptase 5X Reaction Buffer supplied
with this enzyme has a composition of 250mM Tris-HCl (pH 8.3 @ 25°C), 250mM KCl, 50mM MgCl2, 2.5mM spermidine and
50mM DTT.
Enzyme Storage Buffer: AMV Reverse Transcriptase (AMV-RT) is supplied in 200mM potassium phosphate (pH 7.2 @
4°C), 0.2% Triton® X-100, 2mM DTT and 50% glycerol.
*AF9PIM510 1016M510*
AF9PIM510 1016M510
Source: Purified from avian myeloblastosis virus particles.
Storage Conditions: Store at –20°C. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes.
See the expiration date on the Product Information Label.
Unit Definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide
into acid-precipitable material in 10 minutes at 37°C. The reaction conditions are: 50mM Tris-HCl (pH 8.3), 40mM KCl,
8.75mM MgCl2, 10mM DTT, 0.1mg/ml acetylated BSA, 1mM radiolabeled dTTP and 0.25mM poly(A):oligo(dT). See the unit
concentration on the Product Information Label.
Usage Notes:
1. The AMV Reverse Transcriptase 5X Reaction Buffer is intended for use in standard first-strand cDNA synthesis reactions. No
deoxynucleotides are in the buffer; therefore, this buffer must not be substituted for the Promega RiboClone® AMV RT First-
Strand 5X Buffer (Part# C121A), a component of the Universal RiboClone® cDNA Synthesis System (Cat.# C4360), which
does have dNTPs. The Access RT-PCR System (Cat.# A1250) utilizes AMV Reverse Transcriptase and Tfl DNA Polymerase to
provide a combined reverse transcription and PCR without intermediate handling. The reaction buffer provided in the Access Promega Corporation
RT-PCR System is not the same as the 5X Reaction Buffer provided with AMV-RT. The two buffers are not interchangeable. 2800 Woods Hollow Road
Madison, WI 53711-5399 USA
2. The formulation of AMV Reverse Transcriptase 5X Reaction Buffer is not compatible with M-MLV Reverse Transcriptase. Telephone 608-274-4330
3. Up to 10µl of an RT reaction containing AMV-RT and the supplied AMV Reverse Transcriptase Reaction Buffer can be added Toll Free 800-356-9526
to PCR amplification reactions that use Taq DNA Polymerase. If GoTaq® DNA Polymerase (Cat.# M3001) or PCR Master Mix Fax 608-277-2516
(Cat.# M7501) are used, up to 25µl of the RT reaction can be added to a 50µl PCR. Internet www.promega.com

Quality Control Assays PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER

Promega manufactures products for a number of
intended uses. Please refer to the product label for the
intended use statements for specific products.
Activity Assay Promega products contain chemicals which may be
harmful if misused. Due care should be exercised with
First-Strand cDNA Synthesis: First-strand cDNA, of a 1.2kb Control RNA (from Cat.# C4360), is synthesized using 30 all Promega products to prevent direct human contact.
Each Promega product is shipped with documentation
units of AMV Reverse Transcriptase per microgram of template, an oligo(dT) primer and a radiolabeled dNTP. The minimum stating specifications and other technical information.
Promega products are warranted to meet or exceed the
specification is the conversion of >12% of mRNA to cDNA. Full-length cDNA must be observed by gel electrophoresis and stated specifications. Promega's sole obligation and the
autoradiography. customer's sole remedy is limited to replacement of
products free of charge in the event products fail to
perform as warranted. Promega makes no other war-
ranty of any kind whatsoever, and SPECIFICALLY DIS-
Contaminant Activity CLAIMS AND EXCLUDES ALL OTHER WARRANTIES
OF ANY KIND OR NATURE WHATSOEVER, DIRECTLY
Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 25 units of OR INDIRECTLY, EXPRESS OR IMPLIED, INCLUDING,
WITHOUT LIMITATION, AS TO THE SUITABILITY,
AMV Reverse Transcriptase in 50mM Tris (pH 8.3), 40mM KCl, 7mM MgCl2, 10mM DTT for one hour at 37°C. Following PRODUCTIVITY, DURABILITY, FITNESS FOR A PAR-
TICULAR PURPOSE OR USE, MERCHANTABILITY,
incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible CONDITION, OR ANY OTHER MATTER WITH
nicking or cutting. RESPECT TO PROMEGA PRODUCTS. In no event shall
Promega be liable for claims for any other damages,
DNase and RNase Assay: To test for nuclease activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with whether direct, incidental, foreseeable, consequential,
or special (including but not limited to loss of use, rev-
25 units of AMV Reverse Transcriptase in 4mM Tris (pH 8.3), 3.2mM KCl, 0.56mM MgCl2, 0.8mM DTT for one hour at 37°C, enue or profit), whether based upon warranty, contract,
tort (including negligence) or strict liability arising in
and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing connection with the sale or the failure of Promega prod-
specification is <1% release for DNase and <3% release for RNase. ucts to perform in accordance with the stated specifica-
tions.
Physical Purity: AMV Reverse Transcriptase is a 170kDa heterodimer with an α-subunit of 65kDa and a β-subunit of 94kDa.
The purity is >80% in 2 bands (2 subunits) as judged by SDS-polyacrylamide gels with Coomassie® blue staining.

© 1997–2005, 2011, 2016 Promega Corporation. All

Rights Reserved.
GoTaq, RiboClone and RNasin are trademarks of
Promega Corporation and are registered with the U.S.
Patent and Trademark Office.
Coomassie is a registered trademark of Imperial
Chemical Industries. Triton is a registered trademark
of Union Carbide Chemicals and Plastics Co., Inc.
All specifications are subject to change without prior
notice.
Product claims are subject to change. Please contact
Promega Technical Services or access the Promega
online catalog for the most up-to-date information on
Promega products.
Signed by: Part# 9PIM510
R. Wheeler, Quality Assurance Printed in USA. Revised 10/16.
 Usage Information
1. Description 3. Mix gently by flicking the tube and transfer 5µl of the reaction mixture to another tube
containing 2–5µCi [α-32P]dCTP. Do not add label to the remaining 20µl reaction.
AMV Reverse Transcriptase (AMV RT) catalyzes the polymerization of DNA using template
Note: We recommend using [α-32P]dCTP that is less than 1 week old.
DNA, RNA or RNA:DNA hybrids (1). It requires a primer (DNA primers are more efficient
than RNA primers) as well as Mg2+ or Mn2+. The enzyme possesses an intrinsic RNase H 4. Incubate for 60 minutes at 42°C for oligo(dT) primers or at 37°C for random hexamer
activity. Please refer to the Usage Notes, which appear on the other side of this document, primers.
before using this enzyme. 5. Place the reactions, labeled and unlabeled, on ice and add 95µl of 50mM EDTA to
Applications of AMV RT include: the labeled (tracer) reaction. The reaction volume should now total 100µl. The tracer
reaction may be used for an incorporation assay and gel analysis (4).
l First-strand synthesis of cDNA from RNA molecules (2).
l Sequencing of RNA transcripts (3). 6. Perform second-strand synthesis using the unlabeled first-strand reaction (see
references 4 and 5). No phenol extraction or ethanol precipitation is necessary.
2. Standard Applications B. Sequencing of RNA Transcripts
A. First-Strand Synthesis of cDNA A protocol for sequencing RNA transcripts may be found in reference 3.
Reagents to Be Supplied by the User 3. Composition of Buffers and Solutions
l 10mM dNTP mix (Cat.# U1511, U1515 or prepared from 100mM dNTP sets
Cat.# U1240, U1330, U1410, U1420; see Section 3.) dNTP mix
l Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511) 10mM each dATP, dCTP, dGTP and dTTP in water.
l sodium pyrophosphate, 40mM (prewarmed to 42°C) (Prepare from 100mM stock solutions)
l Oligo(dT) (Cat.# C1101) or Random Primers (Cat.# C1181)
l Nuclease-Free Water (Cat.# P1193) 4. References
l EDTA (50mM) 1. Kacian, D.L. (1977) Methods for assaying reverse transcriptase. Meth. Virol. 6, 143.
l [α-32P]dCTP (>400Ci/mmol, 10mCi/ml)
2. Krug, M.S. and Berger, S.L. (1987) First-strand cDNA synthesis primed with oligo(dT).
1. The following procedure (4) uses 2µg of RNA. In a sterile, nuclease-free microcen- Meth. Enzymol. 152, 316–25.
trifuge tube, add the primer to the RNA sample. Use 0.5µg primer/µg RNA in a total
3. Mierendorf, R.C. and Pfeffer, D. (1987) Sequencing of RNA transcripts synthesized
volume of ≤11µl in water. Do not alter the ratio of primer to template RNA. Heat to
in vitro from plasmids containing bacteriophage promoters. Meth. Enzymol. 152, 563–6.
70°C for 5 minutes. Chill the tube on ice for 5 minutes and centrifuge briefly to
collect the solution at the bottom of the tube. 4. Universal RiboClone® cDNA Synthesis System Technical Manual #TM038, Promega
Corporation.
2. Add the following components to the annealed primer/template in the order shown.
5. Sambrook, J. Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory
AMV Reverse Transcriptase 5X Reaction Buffer 5µl
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 8.64.
dNTP mix 2.5µl
RNasin® Ribonuclease Inhibitor 40 units
sodium pyrophosphate, 40mM
(prewarmed to 42°C) 2.5µl
AMV RT 30 units
Nuclease-Free Water to final volume 25µl

Part# 9PIM510
Printed in USA. Revised 10/16.

Promega Corporation · 2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. · Toll Free in the USA 800-356-9526 · Telephone 608-274-4330 · Internet www.promega.com