• My NEB
  • Print
  • PDF
  • M-MuLV Reverse Transcriptase

    Description

    Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase is an RNA-directed DNA polymerase. This enzyme can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. (1-4). M-MuLV Reverse Transcriptase lacks 3´ → 5´ exonuclease activity.

    Highlights

    • Isolated from a recombinant source
    • Synthesizes cDNA from single-stranded RNA or DNA

    Product Source

    The gene encoding M-MuLV Reverse Transcriptase is expressed in E.coli in a vector that results in 16 additional amino acids at the N-terminus and 13 amino acids at the C-terminus. This construct results in a fully functional Reverse Transcriptase protein with a functional RNase H domain.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    M-MuLV Reverse Transcriptase Reaction Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA).oligo(dT) as template primer with 50 mM Tris-HCI (pH 8.3), 6 mM MgCl2, 10 mM dithiothreitol, 0.5 mM [3H]-dTTP and 0.4 mM poly(rA).oligo(dT)12-18.

    Reaction Conditions

    1X M-MuLV Reverse Transcriptase Reaction Buffer
    Incubate at 37°C

    1X M-MuLV Reverse Transcriptase Reaction Buffer:
    50 mM Tris-HCl
    75 mM KCl
    3 mM MgCl2
    10 mM DTT
    pH 8.3 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM Tris-HCl
    150 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% NP-40
    pH 7.6 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 78000 daltons

    Quality Control

    Quality Assurance Statement

    • M-MuLV Reverse Transcriptase is tested for its ability to synthesize full length cDNAs from crude or purified RNA templates. Purified free of detectable levels of RNase, endonuclease and exonuclease activities.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • RNase Activity (2 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    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. Roth, M.J., Tanese, N. and Goff, S.P. (1985). J. Biol. Chem.. 260, 9326-9335.
    4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd Ed.). 5.52-5.55, 8.11-8.17.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Can M-MuLV Reverse Transcriptase be used in other NEBuffers?
    2. Does M-MuLV Reverse Transcriptase require DTT?
    3. Can the M-MuLV Reverse Transcriptase transcribe through RNA-DNA junctions?
    4. Are NEB DNA Polymerases supplied with dNTPs?
    5. How can the length of the product generated by M-MuLV Reverse Transcriptase be increased?
    6. How can the yield be improved when using M-MuLV Reverse Transcriptase?
    7. What are some of the reasons for M-MuLV Reverse Transcriptase reaction failure?
    8. Is M-MuLV Reverse Transcriptase active at temperatures higher than 42°C?
    9. Can DNA be used as a template for M-MuLV Reverse Transcriptase?
    1. First Strand Synthesis Protocol with Reverse Transcriptase

    Selection Tools

    Usage Guidelines & Tips