Certificate of Analysis

M-MLV Reverse Transcriptase:

Part No. Size (units) Part# 9PIM170
M170A 10,000
M170B 50,000 Revised 1/24
Enzyme Storage Buffer: M-MLV Reverse Transcriptase is supplied in 20mM Tris-HCl (pH 7.5), 200mM NaCl,
0.1mM EDTA, 1mM DTT, 0.01% Nonidet® P-40 and 50% glycerol.
M-MLV Reverse Transcriptase 5X Reaction Buffer (M531A): When the M-MLV Reverse Transcriptase 5X Reaction
Buffer supplied with this enzyme is diluted 1:5, it has a composition of 50mM Tris-HCl (pH 8.3 at 25°C), 75mM KCl,
3mM MgCl2 and 10mM DTT.
Source: Purified from an E. coli strain expressing a recombinant clone (1). *AF9PIM170 0124M170*
AF9PIM170 0124M170
Storage Conditions: Store at –30°C to –10°C. Avoid exposure to frequent temperature changes. See the expiration
date on the product 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), 7mM MgCl2, 40mM KCl, 10mM DTT, 0.1mg/ml BSA, 0.5mM [3H]dTTP, 0.025mM oligo(dT),
0.25mM poly(A) and 0.01% NP-40. See the unit concentration on the product label.
Usage Note: M-MLV Reverse Transcriptase is less processive than AMV Reverse Transcriptase, and therefore,
more units of M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction. Thus,
starting with 1μg of mRNA in a first-strand cDNA synthesis, 200 units of the M-MLV enzyme are recommended as
opposed to 25 units of AMV enzyme.

Promega Corporation
2800 Woods Hollow Road
Madison, WI 53711-5399 USA
Telephone 608-274-4330
Toll Free 800-356-9526
Quality Control Assays Fax 608-277-2516
Internet www.promega.com
Activity Assay
First-Strand cDNA Synthesis: Two hundred units of enzyme are used to produce cDNA from 1μg of 1.2kb and
6.5kb control RNAs in separate reactions, using [32P] dCTP as a tracer. The minimum specification is 120ng of PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER
Promega manufactures products for a number of
first-strand cDNA made from 1μg of RNA. The cDNA product must be >90% full length as determined by gel intended uses. Please refer to the product label for the
electrophoresis and autoradiography. intended use statements for specific products.
Promega products contain chemicals which may be
Contaminant Activity harmful if misused. Due care should be exercised with
all Promega products to prevent direct human contact.
DNase and RNase Assay: To test for nuclease activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated Each Promega product is shipped with documentation
with 200 units of M-MLV Reverse Transcriptase in 1X Reaction Buffer for 1 hour at 37°C, and the release of stating specifications and other technical information.
Promega products are warranted to meet or exceed the
radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing stated specifications. Promega’s sole obligation and the
specification is <1% release for both DNase and RNase. customer’s sole remedy is limited to replacement of
products free of charge in the event products fail to
Endonuclease Assay: To test for endonuclease activity, 1μg of Type I supercoiled plasmid DNA is incubated with perform as warranted. Promega makes no other warranty
of any kind whatsoever, and SPECIFICALLY DISCLAIMS
500 units of M-MLV Reverse Transcriptase in 1X Reaction Buffer for 1 hour at 37°C. Following incubation, the AND EXCLUDES ALL OTHER WARRANTIES OF ANY
KIND OR NATURE WHATSOEVER, DIRECTLY OR INDI-
supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking RECTLY, EXPRESS OR IMPLIED, INCLUDING, WITHOUT
or cutting (analysis on 0.4μg of DNA). LIMITATION, AS TO THE SUITABILITY, PRODUCTIVITY,
DURABILITY, FITNESS FOR A PARTICULAR PURPOSE OR
USE, MERCHANTABILITY, CONDITION, OR ANY OTHER
Physical Purity: The purity is >90% as judged by SDS-polyacrylamide gels with Coomassie® blue staining. MATTER WITH RESPECT TO PROMEGA PRODUCTS.
In no event shall Promega be liable for claims for any
other damages, whether direct, incidental, foreseeable,
consequential, or special (including but not limited to
loss of use, revenue or profit), whether based upon
warranty, contract, tort (including negligence) or strict
liability arising in connection with the sale or the failure
of Promega products to perform in accordance with the
stated specifications.

© 1997–2024 Promega Corporation. All Rights Reserved.

RNasin is a registered trademark of Promega
Corporation.
Coomassie is a registered trademark of Imperial
Chemical Industries. Nonidet is a registered trademark of
Shell International Petroleum Company, Ltd.
Products may be covered by pending or issued patents or
may have certain limitations. Please visit our Web site for
more information.
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.
Part# 9PIM170
Signed by:
Printed in USA. Revised 1/24.
R. Wheeler, Quality Assurance
 Usage Information
1. Description 3. Composition of Buffer
Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is an M-MLV RT 5X Reaction Buffer (provided)
RNA-dependent DNA polymerase that can be used in cDNA synthesis with long 250mM Tris-HCl (pH 8.3 at 25°C)
messenger RNA templates (>5kb). M-MLV RT is the preferred reverse transcriptase 375mM KCl
for long mRNA templates because the RNase H activity of M-MLV RT is weaker 15mM MgCl2
than the commonly used Avian Myeloblastosis Virus (AMV) reverse transcriptase.
50mM DTT
Application of M-MLV RT includes:
4. References
• First-strand cDNA synthesis from RNA molecules
Note: M-MLV Reverse Transcriptase is less processive than AMV Reverse 1. Roth, M.J., Tanese, N. and Goff, S.P. (1985) Purification and characterization
Transcriptase, and therefore, more units of the M-MLV enzyme are required to of murine retroviral reverse transcriptase expressed in Escherichia coli.
generate the same amount of cDNA as in the AMV reaction. J. Biol. Chem. 260, 9326–35.
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A
2. First-Strand cDNA Synthesis Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
Materials to Be Supplied by the User New York, 8.64.
(Buffer composition is provided in Section 3.)
• Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511)
• dATP, 10mM (Cat.# U1201, 100mM)
• dCTP, 10mM (Cat.# U1221, 100mM)
• dGTP, 10mM (Cat.# U1211, 100mM)
• dTTP, 10mM (Cat.# U1231, 100mM)
• Nuclease-Free Water (Cat.# P1193)
1. The following procedure uses 2μg of total RNA. In a sterile RNase-free
microcentrifuge tube, add 0.5μg of the primer or primer-adaptor per
microgram of the total RNA sample in a total volume of ≤13.38μl in water.
Heat the tube to 70°C for 5 minutes to melt secondary structure within the
template. Cool the tube immediately on ice to prevent secondary structure
from reforming, then spin briefly to collect the solution at the bottom of the
tube.
2. Add the following components to the annealed primer/template in the order
shown.
Note: Do not alter the ratio of primer to RNA.
M-MLV 5X Reaction Buffer 5.00μl
dATP, 10mM 1.25μl
dCTP, 10mM 1.25μl
dGTP, 10mM 1.25μl
dTTP, 10mM 1.25μl
Recombinant RNasin® Ribonuclease Inhibitor 25 units
M-MLV RT 200 units
Nuclease-Free Water to final volume 25.00μl
3. Mix gently by flicking the tube, and incubate for 60 minutes at 37°C for
random primers or 42°C for other primers or primer-adaptors. The extension
temperature may be optimized between 37°C and 42°C.
4. Perform second-strand synthesis using a protocol of your choice. Standard
protocols for second-strand synthesis may be found in reference 2.
Note: The M-MLV RT Reaction Buffer is compatible with enzymes used in
a number of downstream applications. Phenol extractions and ethanol
precipitations typically are not necessary before performing second-strand
synthesis and amplification.

Part# 9PIM170
Printed in USA. Revised 1/24.
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 · www.promega.com