Description

Thermo Scientific RevertAid Reverse Transcriptase (RT)

is a genetically modified M-MuLV RT. It differs from
wildtype M-MuLV RT by its structure, catalytic properties
PRODUCT INFORMATION and in the optimum activity temperature. The enzyme
Thermo Scientific possesses RNA-dependent and DNA-dependent
polymerase activity and a RNase H activity specific to
RevertAid Reverse RNA in RNA-DNA hybrids which is significantly lower
Transcriptase than that of Avian Myeloblastis Virus (AMV) reverse
transcriptase (1,2).
Pub. No. MAN0012757 RevertAid™ Reverse Transcriptase activity is optimal at
Rev. Date 17 June 2016 (Rev. B.00) 42 °C (active up to 50 °C). The enzyme is capable of first
strand cDNA synthesis up to 13 kb. The enzyme
Lot: _ Expiry Date: _ incorporates modified nucleotides.
Applications
Components #EP0441 #EP0442  First strand cDNA synthesis for RT-PCR and real-time
RT-PCR, see protocol on back page.
RevertAid Reverse  Synthesis of cDNA for cloning and expression.
Transcriptase, 200 10000 U 5  10000 U
 Generation of labeled cDNA probes for microarrays.
U/µL
 DNA labeling (3).
5X Reaction Buffer 1 mL 5  1 mL  Analysis of RNA by primer extension (3).
Source
Store at -20 °C E.coli cells with a cloned fragment of the pol gene
encoding Moloney Murine Leukemia Virus reverse
www.thermofisher.com transcriptase.
For Research Use Only. Not for use in diagnostic procedures.
Definition of Activity Unit CERTIFICATE OF ANALYSIS
One unit of the enzyme incorporates 1 nmol of dTMP into Endodeoxyribonuclease Assay
a polynucleotide fraction in 10 min at 37 °C. No detectable degradation was observed after incubation
Storage Buffer of supercoiled plasmid DNA with RevertAid Reverse
The enzyme is supplied in: 50 mM Tris-HCl (pH 7.5), Transcriptase.
0.1 M NaCl, 1 mM EDTA, 5 mM DTT, Ribonuclease Assay
0.1% (v/v) Triton X-100 and 50% (v/v) glycerol. No detectable degradation was observed after incubation
5X Reaction Buffer of [3H]-RNA with RevertAid Reverse Transcriptase.
250 mM Tris-HCl (pH 8.3 at 25 °C), 250 mM KCl, Labeled Oligonucleotide (LO) Assay
20 mM MgCl2, 50 mM DTT. No detectable degradation after incubation of single-
stranded or double-stranded radiolabeled
Inhibition and Inactivation oligonucleotides with RevertAid Reverse Transcriptase.
Inhibitors: metal chelators, inorganic phosphate, Functional Assay
pyrophosphate and polyamines (2). RevertAid Reverse Transcriptase was tested in synthesis
Inactivated by heating at 70 °C for 10 min. of 1.3 kb first strand cDNA.
Note Quality authorized by: Jurgita Zilinskiene
RevertAid RT has much lower RNase H activity than
Avian Myeloblastosis Virus (AMV) reverse transcriptase.

(continued on back page)

Protocol for First Strand cDNA Synthesis 3. Add the following components in the indicated order:
5X Reaction Buffer 4 µL
The following protocol is optimized to generate first-strand
Thermo Scientific™ RiboLock
cDNA for use in two-step RT-PCR. RNase Inhibitor (#EO0381)
0.5 µL (20 U)
Mix and briefly centrifuge all components after thawing, dNTP Mix, 10 mM each 2 µL
keep on ice. (#R0191) (1 mM final
1. Add into sterile, nuclease-free tube on ice in the indicated concentration)
order: RevertAid Reverse Transcriptase 1 µL (200 U)
Total volume 20 µL
total RNA 0.1 ng-5 µg Mix gently and centrifuge briefly.
or
Template 4. If oligo(dT)18 primer or gene-specific primer is used,
poly(A) RNA 10 pg-500 ng
RNA
or incubate 60 min at 42 °C.
specific RNA 0.01 pg-0.5 µg If random hexamer primer is used, incubate 10 min at
Oligo(dT)18 (#SO131) 0.5 µg (100 pmol) 25 °C followed by 60 min at 42 °C.
or For transcription of GC rich RNA reaction temperature
Primer Random hexamer (#SO142) 0.2 µg (100 pmol) can be increased to 45 °C.
or
5. Terminate the reaction by heating at 70 °C for 10 min.
gene-specific primer 15-20 pmol
Do not heat-inactivate enzyme prior to analysis of long
DEPC-treated water (#R0601) to 12.5 µL cDNA to avoid cleavage.
2. Optional: If RNA template is GC rich or is known to Note
contain secondary structures, mix gently, centrifuge  The reverse transcription reaction product can be directly
briefly and incubate at 65 °C for 5 min, chill on ice, briefly used in PCR or stored at -20 °C.
centrifuge and place on ice.  Use 2 µL of the reaction mix to perform PCR in 50 µL
volume.
References
1. Verma, I.M., Reverse transcriptase, The Enzymes (Boyer, P.D.,
ed), Academic Press Inc., vol. 14, 87-103, 1981.
2. Gerard, G.F. and D'Alessio, J.M., Methods in Molecular Biology,
16, Humana Press, Totowa, N.J., 73-93, 1993. LIMITED USE LABEL LICENSE: Internal Research and Development
Use Only.
3. Sambrook, J., Russell, D.W., Molecular Cloning:
The purchase of this product conveys to the buyer the limited, non-
A Laboratory Manual, the third edition, Cold Spring Harbor exclusive, non-transferable right (without the right to resell, repackage, or
Laboratory Press, Cold Spring Harbor, New York, 2001. further sublicense) to use this product for internal research and
development purposes. No other license is granted to the buyer whether
expressly, by implication, by estoppel or otherwise. In particular, the
purchase of the product does not include or carry any right or license to
use, develop, or otherwise exploit this product commercially and no
rights are conveyed to the buyer to use the product or components of the
product for purposes including but not limited to provision of services to a
third party, generation of commercial databases or clinical diagnostics.
This product is sold pursuant to authorization from Thermo Fisher
Scientific and Thermo Fisher Scientific reserves all other rights. For
information on purchasing a license for uses other than internal research
and development purposes, please contact [email protected] or
Out Licensing, Life Technologies Inc., 5791 Van Allen Way, Carlsbad,
California 92008.

PRODUCT USE LIMITATION

This product is developed, designed and sold exclusively for research
purposes and in vitro use only. The product was not tested for use in
diagnostics or for drug development, nor is it suitable for administration
to humans or animals.
Please refer to www.thermofisher.com for Material Safety Data Sheet of
the product.

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