M-MLV Reverse Transcriptase
Code
Description
Size
1B1414-200RXN
M-MLV Reverse Transcriptase
Includes:
M-MLV RT, M-MLV RT Buffer,
5X, & DTT Solution, 0.1M
1 x 62.5 µL of M-MLV RT
(40KU)
1B1414-1000RXN
1 x 1 mL of M-MLV RT
(200KU)
General Information:
Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is a high
purity, recombinant RNA-directed DNA polymerase used to synthesize first-strand
complementary DNA from a primer and single-strand RNA template. Unlike Avian
Myeloblastosis Virus (AMV) reverse transcriptase, M-MLV RT has a weaker
intrinsic RNase H activity and is not inhibited by ribosomal or transfer RNA. It is
therefore useful for synthesis of long, full length cDNA (up to ~6kb) from mRNA in
total RNA preparations.
Storage/Stability:
Store at -20°C. Ready One-Step RT PCR Mix, 2X is s table through 15 freeze-thaw
cycles.
Application Disclaimer
For research use only.
Not for therapeutic or diagnostic use.
________________________________________________________________________________
1-800-448-4442
www.amresco-inc.com
Page 1 of 3
Procedure
1. Thaw all components (except enzyme), mix
thoroughly, and centrifuge before use. Hold on ice
before use.
2. Add the following to a 0.2-mL thin-walled PCR tube
or 96-well PCR reaction plate sitting on ice:
Component
Volume (µL)
RNA (up to 5 µg total RNA)
variable
Nuclease-free water
variable
10 mM dNTP mix
1.0
Primer solution
2.0
Final Volume
10.0
**Note: For a mixed primer strategy use 2 µL of random primer
plus 2 µL of oligo dT.**
3. Mix components by gently vortexing and then
centrifuge 10 sec to collect contents.
4. Incubate for 5 min at 65°C and then immediately
place on ice.
5. Add the following components to the primed RNA
template mixture:
Component
Volume (µL)
primed RNA template mixture
10.0
Nuclease-free water
2.0
M-MLV RT Buffer, 5X
4.0
DTT Solution, 0.1M
2.0
RNase inhibitor protein, 40U/µL
1.0
(optional)
M-MLV RT, 200U/µL
1.0
Final Volume
20.0
**Note: For multiple first-strand reactions, prepare a master mix
with the M-MLV RT Buffer, 5X, DTT Solution, 0.1 M, RNase
inhibitor protein, and RT and dispense 10 µL into each tube.**
6. Mix components by gently vortexing and then
centrifuge 10 sec to collect contents.
7. Incubate reactions for:
Oligo(dT)20 Primer or
Random Primers
Gene-Specific Primer
10 min at 25°C
45 min at 42ºC
60 min at 42ºC
5 min at 85ºC
5 min at 85ºC
Hold at 4ºC
Hold at 4ºC
**Note: For a mixed primer strategy (Random Primer plus Oligo
dT), use the random primer incubation protocol. It is critical to
allow random primers to anneal at 20 to 25ºC before increasing
the incubation temperature to 42°C.**
8. After completion of cDNA synthesis, use 1/5th to
1/10th of the first-strand reaction (2-4 µL) for PCR
amplification. Dilution of the first-strand reaction with
TE buffer [10 mM Tris (pH 8.0), 0.1 mM EDTA] is
useful for addition of larger volumes (5 – 10 µL) to
the PCR, or when storing the material for future use.
cDNA may be stored at -20ºC.
Frequently Asked Questions
Questions
Answers
Why does the first-strand 1. If random primers were used,
synthesis product
random sizes of first-strand
appear as a smear on an
synthesis can be expected
agarose gel
2. M-MLV RT is not processive
enough to completely
reverse transcribe RNA
lengths >6000 nucleotides
Why do I not see any
1. Residual DNase
DNA product after
contamination from DNaseamplification of cDNA on
treated template RNA
my gel?
2. RNase contamination in your
first-strand synthesis reaction
3. The yield of the product is
too low to be observed.
Why do I see multiple or
1. Contamination of another
unexpected product
target RNA or DNA
sizes from amplification
2. Incomplete digestion of
of cDNA?
genomic DNA
3. Multiple RNA target
sequences exist
4. Non-specific primer
annealing
5. Primer dimers
Related Products
Code
0649-50KU
0649-100KU
Product
DNase I
E633-2KU
E633-10KU
Ribonuclease Inhibitor (RNase
Inhibitor)
E891-100ML
E891-500ML
E891-50ML-PUMP
E891-100ML-PUMP
E891-25PK
NucleasEliminator™
N580-30ML
N580-100ML
N580-200ML
N643-KIT
N788-KIT
NucleasEliminator Wipes™
Ribozol™ RNA Extraction
Reagent
Ribozol™ Plus RNA Purification
Kit
Phenol-Free Total RNA
Purification Kit
________________________________________________________________________________
1-800-448-4442
www.amresco-inc.com
Page 2 of 3
ZY0539 Rev 0. 01/2012 ©Copyright 2010 by
AMRESCO, LLC. All Rights Reserved. AMRESCO® is a
registered trademark of AMRESCO, LLC.
Corporate Headquarters
AMRESCO, LLC.
6681 Cochran Road
Solon, OH 44139 USA
Tel: (440) 349-1199
Toll free: 1-800-448-4442 (US & Canada)
Fax: (440) 349-2441
e-mail: info@amresco-inc.com
To Order (USA & Canada)
www.amresco-inc.com
Toll free: 1-800-448-4442
Toll free fax: 1-800-326-3733
Fax: (440) 349-3255
e-mail: dom-sales@amresco-inc.com
International Orders:
Tel: (440) 349-1199
Fax: (440) 349-2441
e-mail: int-sales@amresco-inc.com
Technical Support
Toll free: 1-800-610-2789 (USA & Canada)
Fax: (440) 349-0235
e-mail: techinquiry@amresco-inc.com
________________________________________________________________________________
1-800-448-4442
www.amresco-inc.com
Page 3 of 3