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  • AMV Reverse Transcriptase

    Description

    Avian Myeloblastosis Vi­rus (AMV) Reverse Transcriptase is an RNA-directed DNA polymerase. This en­zyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template (1-3).

    Highlights

    • Synthesizes cDNA from single-stranded RNA

    Product Source

    Avian Myeloblastosis Vi­rus (AMV)

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    AMV Reverse Transcriptase Reaction Buffer10X

    Advantages and Features

    Applications

    • cDNA Synthesis
    • RNA Sequencing
    • RT-PCR

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into an acid-insoluble form in 10 minutes at 37°C using poly(rA)-oligo(dT) as template primer.

    Reaction Conditions

    1X AMV Reverse Transcriptase Reaction Buffer
    Incubate at 37°C

    1X AMV Reverse Transcriptase Reaction Buffer:
    50 mM Tris-HCl
    75 mM Potassium Acetate
    8 mM Magnesium Acetate
    10 mM DTT
    pH 8.3 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    0.2 mM potassium phosphate
    2 mM DTT
    50% Glycerol
    0.2% Triton® X-100
    pH 7.2 @ 25°C

    Unit Assay Conditions

    75 mM potassium acetate, 50 mM Tris-HCl (pH 8.3) 8 mM magnesium acetate, 0.5 mM [3H]-dTTP, 0.2 mM poly(rA)•oligo(dT)12–18 and 10 mM DTT.

    Notes

    1. Reaction Conditions: Incubate at 37-52°C.
    2. The yield and size of cDNA transcript increases with increasing amounts of RT.
    3. Storage: Once thawed, store at -20°C. Repeated freeze thaw cycles will inactivate the enzyme. Aliquots can be stored for longer periods at -70°C.

    References

    1. Krug, M.S. et al. (1987). Meth. Enzymol. 152
    2. Sambrook.J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 8-64. Cold Spring Harbor Laboratory Press.
    3. Kacian, D.L. (1977). Meth. Virol.. 6, 143.

    Protocols

    1. First Strand Synthesis Protocol with AMV Reverse Transcriptase (M0277)

    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.

    Selection Tools

    Usage Guidelines & Tips

    Interactive Tools

    Quality Control

    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.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • 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.
    • RNase Activity (2 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    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.