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  • EpiMark® Hot Start Taq DNA Polymerase

    Description

    EpiMark Hot Start Taq DNA Polymerase is a mixture of Taq DNA Polymerase and a temperature sensitive, aptamer-based inhibitor. The inhibitor binds reversibly to the enzyme, inhibiting polymerase activity at temperatures below 45°C, but releases the enzyme during normal PCR cycling conditions. This permits assembly of reactions at room temperature. An advantage of the aptamer-based hot start mechanism is that it does not require a separate high temperature incubation step to activate the enzyme. The advanced aptamer-based hot-start activity coupled with the supplied optimized reaction buffer makes the EpiMark Hot Start Taq DNA Polymerase an excellent choice for use on bisulfite-converted DNA.

    Taq
    DNA polymerase possesses a 5´- 3´ polymerase activity (1,2,3) and a 5´ flap endonuclease activity (4,5).

    EpiMark Hot Start Taq DNA Polymerase is supplied with 5X EpiMark Hot Start Taq Reaction Buffer, providing robust amplification for bisulfite-converted DNA and AT-rich amplicons.

    Product Source

    An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    EpiMark® Hot Start Taq Reaction Buffer Pack-205X

    Advantages and Features

    Applications

    • PCR from bisulfite-converted DNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 15 nmol dNTP into acid insoluble material in 30 minutes at 75°C.

    Reaction Conditions

    1X EpiMark® Hot Start Taq Reaction Buffer Pack

    1X EpiMark® Hot Start Taq Reaction Buffer Pack:
    20 mM Tris-HCl
    1.8 mM MgCl2
    22 mM KCl
    0.06% IGEPAL® CA-630
    0.05% Tween® 20
    22 mM (NH4)2SO4
    pH 8.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    0.5% Tween® 20
    0.5% IGEPAL® CA-630
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    Molecular Weight

    Theoretical: 94000 daltons

    5' - 3' Exonuclease

    Yes

    3' - 5' Exonuclease

    No

    Resulting Ends

    • Single-base 3´ Overhangs

    Unit Assay Conditions

    1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 200 µg/ml activated Calf Thymus DNA.

    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.
    • Inhibition of Primer Extension (Hot Start, Radioactivity Incorporation):
      The hot start polymerase is compared to the polymerase in non-hot start conditions using primer extension; inhibition is assessed by comparing radioactive incorporation.
    • PCR Amplification (Hot Start):

      The polymerase is tested in a hot start polymerase chain reaction (PCR) using Lambda DNA as the control template, specific primers and human genomic DNA, resulting in an increase in yield of the expected Lambda product and a decrease in non-specific genomic bands when compared to a non-hot start control reaction.

    • Single Stranded DNase Activity (FAM Labeled Oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.

    References

    1. Chien, A., Edgar, D.B. and Trela, J.M. (1976). J. Bact. . 127, 1550-1557.
    2. Kaledin, A.S., Slyusarenko, A.G. and Gorodetskii, S.I. (1980). Biokhimiya . 45, 644-651.
    3. Lawyer, F.C. et al. (1993). PCR Methods And Appl.. 2, 275-287.
    4. Longley, M.J., Bennett, S.E. and Mosbaugh D.W. (1990). Nucleic Acids Res. . 18, 7317-7322.
    5. Lyamichev, V., Brow, M.A. and Dahlberg, J.E. (1993). Science. 260, 778-783.
    6. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
    7. Powell, L.M. et al. (1987). Cell. 50, 831-840.
    8. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques . 15, 372-374.

    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. Does EpiMark® Hot Start Taq DNA Polymerase require an extended initial incubation to activate the polymerase?
    2. How has this product been optimized for use with bisulfite-converted DNA?
    3. Can EpiMark® Hot Start Taq DNA Polymerase be used in any other reaction buffers?
    4. What type of PCR ends are produced with EpiMark® Hot Start Taq DNA Polymerase?
    1. EpiMark® Hot Start Taq DNA Polymerase Guidelines for PCR (M0490)