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  • LongAmp™ Hot Start Taq 2X Master Mix

    Description

    LongAmp Hot Start Taq 2X Master Mix contains a unique blend of aptamer-based Hot Start Taq and Deep VentR™ DNA Polymerases. The aptamer-based 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 PCR reactions at room temperature. The LongAmp® Hot Start Taq 2X Master Mix does not require a separate high temperature incubation step to activate the enzyme. The 3´→5´ exonuclease activity of Deep VentR DNA Polymerase increases the fidelity and robust amplification of Hot Start Taq DNA Polymerase (1). LongAmp® Hot Start Taq DNA Polymerase offers two-fold higher fidelity than Hot Start Taq DNA Polymerase alone. The convenient master mix formulation is supplied at a 2X concentration and contains dNTPs and Mg++, requiring only the addition of primers and DNA template for robust amplification. A wide range of PCR products can be generated; up to 30 kb from lambda or human genomic DNA.

    Amplification of a 8 kb amplicon from varying amounts of Jurkat genomic DNA using LongAmp Hot Start Taq 2X Master Mix. Starting template amounts are indicated below the gel. Marker M is the NEB 2-Log DNA Ladder (NEB #N3200 ).

    Product Source

    An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1 and an E. coli strain that carries the Deep VentR DNA Polymerase gene from Pyrococcus species GB-D.

    Unit Definition:
    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 75°C.

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

    Advantages and Features

    Applications

    • High-specificity Long Range PCR 
    • High-throughput PCR
    • Colony PCR

    Properties and Usage

    Buffer Composition

    60 mM Tris-SO4
    20 mM (NH4)2SO4
    2 mM MgSO4
    3% Glycerol
    0.06% IGEPAL® CA-630
    0.05% Tween® 20
    0.3 mM dNTPs
    125 units/ml LongAmp® Hot Start Taq DNA Polymerase
    pH 9@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.
    • 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 (DNA Polymerase):

      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.

    • 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.

    Notes

    1. Product specifications for individual components in the LongAmp Hot Start Taq 2X Master Mix are available separately.

    References

    1. Barnes, W.M. (1994). Proc. Natl. Acad. Sci. USA. 91, 1350-1354.
    2. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
    3. Powell, L.M. et al (1987). Cell. 50, 831-840.
    4. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
    5. 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. What is the fidelity of the LongAmp™ Hot Start Taq 2X Master Mix compared to Taq DNA Polymerase?
    2. Can the extension step be carried out at 72°C when using LongAmp™ Hot Start Taq 2X Master Mix?
    3. What is the extension rate when using LongAmp™ Hot Start Taq 2X Master Mix?
    4. What type of DNA ends result from a primer extension reaction or a PCR using LongAmp™ Hot Start Taq 2X Master Mix?
    5. Why is the product a smear when visualized on an agarose gel?
    6. Can LongAmp™ Hot Start Taq 2X Master Mix be used to amplify GC-rich amplicons?
    1. Protocol for LongAmp™ Hot Start Taq 2X Master Mix

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Using an extension temperature of 65°C is recommended in LongAmp Hot Start Taq reactions.