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    Hot Start Taq DNA Polymerase

    Description

    Hot Start Taq DNA Polymerase is a mixture of Taq DNA Polymerase and an aptamer-based inhibitor. The inhibitor binds reversibly to the enzyme, inhibiting polymerase activity at temperatures below 45°C, but releases the enzyme during normal cycling conditions, allowing reactions to be set up at room temperature. This aptamer-based hot start does not require a separate high temperature incubation step to activate the enzyme. Taq DNA polymerase possesses a 5´→3´ polymerase activity (1)(2)(3)and a 5´ flap endonuclease activity(4)(5).

    It is supplied with 10X Standard Taq Reaction Buffer, which is detergent-free and designed to be compatible with existing assay systems.

    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
    Standard Taq Reaction Buffer Pack-2010X

    Advantages and Features

    Applications

    • High-specificity PCR
    • Routine PCR
    • Microarray Analysis
    • Colony PCR

    Properties and Usage

    Unit Definition

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

    Reaction Conditions

    1X Standard Taq Reaction Buffer Pack

    1X Standard Taq Reaction Buffer Pack:
    10 mM Tris-HCl
    50 mM KCl
    1.5 mM MgCl2
    pH 8.3 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    1X stabilizers
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    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 DNA Binding (FAM labeled oligo):
      The product is tested for its ability to produce a mobility shift when incubated using standard reaction conditions with a single stranded FAM labeled oligo.

    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. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
    7. Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res. 18, 7465.

    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 Hot Start Taq DNA Polymerase require an extended initial incubation to activate the polymerase?
    2. How should I set up a PCR using Hot Start Taq DNA Polymerase?
    3. Can Hot Start Taq DNA Polymerase be used in other buffers?
    4. What type of DNA ends result from a primer extension reaction or a PCR reaction using Hot Start Taq DNA Polymerase?
    5. When should Hot Start Taq DNA Polymerase be used in a primer extension reaction or for PCR?
    6. Will the 5'→3' flap endonuclease activity of Hot Start Taq DNA Polymerase degrade primers?
    7. Can Hot Start Taq DNA Polymerase be used for nick translation?
    8. How should I determine an appropriate annealing temperature for my reaction?
    9. What is the longest amplicon that can be obtained with Hot Start Taq DNA Polymerase?
    10. Can Hot Start Taq DNA Polymerase be used with uracil-containing primers or bisulfite-treated DNA?
    1. PCR Using Hot Start Taq DNA Polymerase (M0495)
    2. A-Tailing with Taq Polymerase