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    LongAmp® Hot Start Taq DNA Polymerase

    Description

    LongAmp Hot Start Taq DNA Polymerase is 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. 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 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. A wide range of PCR products can be generated; up to 30 kb from lambda or human genomic DNA.

    Amplification of a 6 kb amplicon from varying amounts of human genomic DNA using LongAmp Hot Start Taq DNA Polymerase. 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.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    LongAmp® Taq Reaction Buffer Pack-205X

    Advantages and Features

    Features

    • Room temperature reaction setup
    • More robust & longer amplicons than Taq

    Applications

    • High-specificity Long Range PCR
    • Colony PCR

    Properties and Usage

    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.

    Reaction Conditions

    1X LongAmp Taq Reaction Buffer

    1X LongAmp™ Taq Reaction Buffer:
    60 mM Tris-SO4
    20 mM (NH4)2SO4
    3% Glycerol
    0.06% IGEPAL® CA-630
    0.05% Tween® 20
    2 mM MgSO4
    pH 9.1 @ 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

    5' - 3' Exonuclease

    Yes

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    +1

    Unit Assay Conditions

    1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 15 nM primed M13 DNA.

    Notes

    1. 5'→3' flap endonuclease destroys displaced strand.

    References

    1. Barnes, W.M. (1994). Proc. Natl. Acad. Sci. USA. 91, 2216-2220.
    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.

    FAQs

    1. What is the recommended enzyme amount when using LongAmp™ Hot Start Taq DNA Polymerase?
    2. What is the fidelity of the LongAmp™ Hot Start Taq DNA Polymerase compared to Taq DNA Polymerase?
    3. Can the extension step be carried out at 72°C when using LongAmp™ Hot Start Taq DNA Polymerase?
    4. What type of DNA ends result from a primer extension reaction or a PCR using LongAmp™ Hot Start Taq DNA Polymerase?
    5. What is the extension rate when using LongAmp™ Hot Start Taq DNA Polymerase?
    6. Why is the product a smear when visualized on an agarose gel?
    7. Can LongAmp™ Hot Start Taq DNA Polymerase be used to amplify GC-rich amplicons?

    Tech Tips

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

    Protocols

    1. PCR Protocol for LongAmp® Hot Start Taq DNA Polymerase (M0534)

    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 Charts

    Usage Guidelines & Tips

    Troubleshooting Guides

    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. Further information regarding NEB product quality can be found here.
    • 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 is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.
    • PCR Amplification (Hot Start, Human Genomic DNA):
      The polymerase is tested in a hot start polymerase chain reaction (PCR) using Human genomic DNA as the control template and specific primers, resulting in an increase in yield of the expected product and a decrease in non-specific genomic bands when compared to a non-hot start control reaction.

    Certificate of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    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.