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  • OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer

    Description

    OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer is an optimized, ready-to-use blend of Taq and Deep VentR™ DNA Polymerases combined with an aptamer-based inhibitor. This enzyme blend is ideally suited to PCR applications from GC-rich templates, including pure DNA solutions, bacterial colonies, and cDNA products. The 3´→5´ exonuclease activity of Deep Vent DNA Polymerase increases the fidelity and robust amplification of Taq DNA Polymerase (1). The hot start nature of the enzyme offers convenience with decreased interference from primer dimers and secondary products. The convenient master mix formulation contains dNTPs, MgSO4, buffer components and stabilizers as well as two commonly used tracking dyes for DNA gels. On a 1% agarose gel in 1X TBE, Xylene Cyanol FF migrates at ~4 kb and Tartrazine migrates at ~10 bp. Both dyes are present in concentrations that do not mask co-migrating DNA bands.

    Amplification of a selection of sequences with varying GC content from human genomic DNA using OneTaq Hot Start DNA Polymerase. The presence or absense of an extended room temperature incubation does not affect performance. Ampicon size is indicated next to gel and GC content is indicated below gel. Marker M is the 1 kb DNA Ladder.

    Highlights


    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
    OneTaq High GC Enhancer10X

    Advantages and Features

    Applications

    • GC-rich PCR
    • High Sensitivity PCR
    • High Throughput PCR
    • Colony PCR
    • Long PCR (up to ~6 kb genomic)

    Properties and Usage

    Storage Temperature

    -20°C

    Buffer Composition

    80 mM Tris-SO4
    20 mM (NH4)2SO4
    0.2 mM dNTPs
    2 mM MgSO4
    5% Glycerol
    5% DMSO
    0.06% IGEPAL® CA-630
    0.05% Tween® 20
    1X Xylene Cyanol
    1X Tartrazine
    25 units/ml OneTaq Hot Start DNA Polymerase
    pH 9.2@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.

    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 OneTaq Hot Start Quick-Load Master Mix with GC Buffer, DNA template and primers in a total reaction volume of 50 µl.

    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.
    • PCR Amplification (Buffer Dependent, GC-rich, Master Mix)  :
      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a GC-rich control template and specific primers, resulting in the buffer-dependent production of the expected product.
    • PCR Amplification (Enhancer Dependent, GC-rich, Master Mix)  :
      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a GC-rich control template and specific primers, resulting in the enhancer-dependent production of the expected product.

    Notes

    1. Product specifications for individual components in the OneTaq Hot Start Quick-Load 2X Master Mix with GC Buffer are available separately.
    2. OneTaq Hot Start Quick-Load 2X Master Mix with GC Buffer is stable for fifteen freeze-thaw cycles when stored at -20°C.
    3. OneTaq Hot Start Quick-Load 2X Master Mix with GC Buffer is also stable for one month at 4°C, so for frequent use, an aliquot may be kept at 4°C.

    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.

    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. How should I set up a PCR using the OneTaq® Hot Start Master Mixes?
    2. Can I use my regular Taq-based cycling conditions for OneTaq® Hot Start DNA Polymerase based products?
    3. How do I activate OneTaq® Hot Start Polymerase?
    4. What are the stability and storage requirements of the Quick-Load® OneTaq® Master Mixes?
    5. What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?
    6. How long a product can be made by OneTaq® DNA Polymerase?
    7. Can OneTaq® DNA Polymerase be used with uracil-containing primers or bisulfite-treated DNA?
    8. What is the fidelity of OneTaq® DNA Polymerase?
    9. When should I add the High GC Enhancer?
    10. Where can I find help troubleshooting my PCR?
    11. How should I determine an appropriate annealing temperature for my reaction?
    12. How is OneTaq DNA Polymerase different from LongAmp™ Taq DNA Polymerase?
    1. Protocol for OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer
    2. Guidelines for PCR Optimization with OneTaq® and OneTaq® Hot Start DNA Polymerases

    Usage Guidelines & Tips

    Interactive Tools

    Application Notes

    A master mix with a gel loading dye is the ultimate time-saver! It allows for easy reaction set up and with the gel loading dye already in the mix, the results of your PCR are available even faster.