M-MLV Reverse Transcriptase

M-MLV Reverse Transcriptase
Description

M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase is a an RNA-directed DNA polymerase that can synthesize a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid.
 
Name Cat# Size Concentration Price Qty
MMLV Reverse Transcriptase
SSI-100
10,000U
200 U/μl
$45.00
MMLV Reverse Transcriptase
SSI-200
50,000U
200 U/μl
$135.00
MMLV Reverse Transcriptase
SSI-300
100,000U
200 U/μl
$225.00

Description:
M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase is a an RNA-directed DNA polymerase that can synthesize a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. Unlike other reverse transcriptase such as AMV (Avian Myeloblastosis Virus) RT, M-MLV RT lacks endonuclease activity and the RNase H activity is also lower. This recombinant enzyme is isolated from an E.coli strain and it is ideal for first-strand cDNA synthesis from RNA template, 5'-RACE, primer extension, RT-PCR and qRT-PCR.

Catalog No.                 
SSI-100, SSI-200 and SSI-300
Source                         
E.coli

Concentration          
200 u/μl

Storage Buffer         
20 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.05% (v/v)Triton X-100, 0.1 mM       EDTA, 0.1 M NaCl and  50% (v/v) glycerol.

Reaction Buffer (5x)
250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, and 50 mM DTT
Unit Definition     
One unit of the enzyme incorporates I nmole of dTTP into acid-precipitable material in 10 minutes at 37˚C using poly (A): oligo (dT)25 as template-primer
Quality Control     
This enzyme has passed the quality control assays: SDS-PAGE analysis for purity, functional absence of endonuclease activities, functional absence of exonuclease activities, functional absence of protease activity

Protocol
First-Strand cDNA Synthesis
Materials to Be Supplied by the User

  • RNAse Inhibitor (Cat.# RNIN-100, RNIN-200, RNIN-300)
  • dNTP, 10mM (Cat.# dNTP-10M, dNTP-25M)
  • Nuclease-Free Water

The following procedure uses 10 pg to 5 µg of total RNA or 10 pg to 500 ng of mRNA.

  1. In a sterile RNase-free microcentrifuge tube, add primers (200-500 ng of oligo (dT)12-18, 50-250 ng of random primer or 2 pmol of specific primers). Heat the tube to 70°C for 5 minutes spin briefly and incubate on ice for 1 min to denature any possible secondary structure within the template. 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 mRNA.
    5 µl 5X Reaction Buffer; 5 µl of 10mM dNTP mixture (10mM each dATP, dGTP, dCTP and dTTP)
    25 units RNAse Inhibitor
    0.5 µl M-MLV Reverse Transcriptase (200 u/ μl)
    Add nuclease-free water to final volume 25µl
  3. Mix gently. For random primers, incubate tube at 25°C for 5 min. Perform first-strand synthesis at 42°C for 60 min.
  4. Inactivate the enzyme by incubation at 90°C for 10 min after reaction.
    Note: The 5X Reaction Buffer is compatible with enzymes used in a number of downstream applications. Typically there is no need for phenol extractions or ethanol precipitations using this protocol before any PCR amplification.
Storage and Handling      -20˚C
References
  1. Verma, I.M. (1975). J. Virol.. 15, 843-854.
  2. Gerard, G.F. and Grandgenett, D.P. (1975). J. Virol.. 15, 785-797.
  3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A; Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 8.64.

Manual & Protocols



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