• My NEB
  • Print
  • PDF
  • OneTaq® Hot Start DNA Polymerase

    Description

    OneTaq® Hot Start DNA Polymerase is an optimized blend of Taq and Deep VentR™ DNA polymerases combined with an aptamer-based inhibitor. The 3´→ 5´ exonuclease activity of Deep Vent DNA Polymerase increases the fidelity and robust amplification of Taq DNA Polymerase (1) and the hot start formulation combines convenience with decreased interference from primer dimers and secondary products. The OneTaq Reaction Buffers and High GC Enhancer have been formulated for robust yields with minimal optimization, regardless of a template’s GC content.

    The inhibitor binds reversibly, blocking polymerase activity at temperatures below 45°C. The polymerase is activated during normal cycling conditions, allowing reactions to be set up at room temperature. OneTaq Hot Start DNA Polymerase does not require a separate high temperature incubation step to activate the enzyme and can be used in typical Taq-based cycling protocols.

    OneTaq Hot Start DNA Polymerase is supplied with two 5X buffers: (Standard and GC), as well as a High GC Enhancer solution. For most routine and/or AT-rich amplicons (Lambda, etc.) or complex amplicons with up to ~65% GC content, OneTaq Standard Reaction Buffer provides robust amplification. For GC-rich amplicons, the OneTaq GC Reaction Buffer can improve both performance and yield. For particularly high GC or difficult amplicons (i.e. ≥ 75% GC), the OneTaq High GC Enhancer can be added at a final concentration of 10–20% to reactions containing OneTaq GC Reaction Buffer.

    Amplification of a selection of sequences with varying GC content from human and C. elegans genomic DNA using OneTaq Hot Start DNA Polymerase. The presence or absence of an extended room temperature incubation does not affect performance. GC content is indicated below gel. Marker M is the 1 kb DNA Ladder (NEB #N3232 ).

    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® Standard Reaction Buffer-205X
    OneTaq® GC Reaction Buffer-205X
    OneTaq High GC Enhancer100%

    Advantages and Features

    Features

    • Room temperature reaction setup

    Applications

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

    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 OneTaq® Standard Reaction Buffer

    1X OneTaq® Standard Reaction Buffer Pack:
    20 mM Tris-HCl
    22 mM NH4Cl
    22 mM KCl
    1.8 mM MgCl2
    0.06% IGEPAL® CA-630
    0.05% Tween® 20
    pH 8.9 @ 25°C

    Usage Concentration

    1.25 units/50µl reaction

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    No

    5' - 3' Exonuclease

    Yes

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    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 (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 (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 (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.
    • 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. The OneTaq High GC Enhancer should not be used alone. It should be added only to the OneTaq GC Reaction Buffer and will typically enhance yields when other conditions have failed.
    2. Product specifications for individual components in the OneTaq Hot Start DNA Polymerase mix are available separately.

    References

    1. Barnes, W.M. (1994). Proc. Natl. Acad. Sci. USA. 91, 2216-2220.
    2. Saiki R.K. et al. (1985). Science. 230, 1530-1554.
    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 OneTaq® Hot Start DNA Polymerase?
    2. Can I use my regular Taq-based cycling conditions for OneTaq® DNA Polymerase based products?
    3. Which buffer should I use?
    4. How do I activate OneTaq® Hot Start Polymerase?
    5. Is OneTaq® Hot Start DNA Polymerase active in other Taq reaction buffers?
    6. When should I add the High GC Enhancer?
    7. Can OneTaq® DNA Polymerase be used in colony PCR?
    8. What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?
    9. How long a product can be made by OneTaq® DNA Polymerase?
    10. Can OneTaq® DNA Polymerase be used with uracil-containing primers or bisulfite-treated DNA?
    11. What is the fidelity of OneTaq® DNA Polymerase?
    12. Which buffer should I use if I want to control the level of magnesium in the reaction?
    13. Where can I find help troubleshooting my PCR?
    14. How should I determine an appropriate annealing temperature for my reaction?
    15. How is OneTaq DNA Polymerase different from LongAmp™ Taq DNA Polymerase?
    1. Protocol for OneTaq Hot Start DNA Polymerase (M0481)

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Application Notes

    Citations

    • Gorski L, Walker S, Liang AS, Nguyen KM, Govoni J, et al. (2014) Comparison of Subtypes of Listeria monocytogenes Isolates from Naturally Contaminated Watershed Samples with and without a Selective Secondary Enrichment PLoS ONE 9(3), e92467. PubMedID: 24651315, DOI: 10.1371/journal.pone.0092467
    Did you know most OneTaq Hot Start reactions amplify more efficiently and robustly when you use a 68°C extension temperature?