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  • Phusion® High-Fidelity PCR Kit

    Description

    High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Manufactured and quality-controlled at New England Biolabs, Thermo Scientific® Phusion High-Fidelity DNA Polymerase offers both high-fidelity and robust performance, and thus can be used for all PCR applifications. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate >50-fold lower than of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase (7), Phusion is one of the most accurate thermostable polymerases available. Phusion DNA Polymerase possesses 5'→3' polymerase activity, 3'→5' exonuclease activity and will generate blunt-ended products.

    The Phusion High-Fidelity PCR Kit contains a sufficient supply of Phusion High-Fidelity DNA Polymerase, Phusion HF and GC Buffers, deoxynucleotides, magnesium chloride, DMSO, and DNA size standard to perform 50 reactions (small) or 200 reactions (large). Control template and primers are provided for 20 control reactions.

    Notice to Customers:

    Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is now manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. Further details on this change can be found in the following customer communication. However, it is important to note that the catalog numbers for these products have changed. For any questions, please use our Phusion Request for Technical Support or call NEB Technical Support at 1-800-632-7799.

    Phusion® is a registered trademark and property of Thermo Fisher Scientific.

    Fidelity assays were performed using a lacI-based method modified from Frey & Suppman, 1995.

    A 3.8 kb fragment was amplified from 50 ng of Jurkat gDNA using different polymerases. Reactions were carried out according to the manufacturer's recommended conditions (cycling conditions are shown below). Extension times are indicated (in minutes). Ladder L is a 1 kb DNA Ladder .

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Deoxynucleotide (dNTP) Solution Mix-2010 mM
    Control Lambda Template0.5 μg/ml
    1.3 kb Control Primer Mix4 μM
    10 kb Control Primer Mix4 μM
    Phusion® High-Fidelity DNA Polymerase-202,000 units/ml
    Quick-Load DNA Marker, Broad Range100 μg/ml
    DMSO100%
    Phusion® HF Buffer-205X
    Phusion® GC Buffer Pack-205X
    MgCl2 solution50 mM

    Advantages and Features

    Features

    Application features:
    • PCR
    • Primer Extension
    • Cloning
    • Longer Difficult Amplification
    • High-Throughput PCR

    Properties and Usage

    Storage Temperature

    -20°C

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

    References

    1. Saiki, R.K. et al. (1985). Science. 230, 1350-1354.
    2. Chien, A., Edgar, D.B. and Trela, J.M. (1976). J. Bact.. 127, 1550-1557.
    3. Kaledin, A.S., Slyusarenko, A.G. and Gorodetskii, S.I. (1980). Biokhimiya. 45, 644-651.
    4. Lawyer, F.C. et al. (1993). PCR Method and Appl.. 2, 275-287.
    5. Longley, M.J., Bennett, S.E. and Mosbaugh D.W. (1990). Nucleic Acids Res.. 18, 7317-7322.
    6. Lyamichev, V., Brow, M.A. and Dahlberg, J.E. (1993). Science. 260, 778-783.
    7. Frey and Suppman (1995). Biochimica. 34-35.
    8. Breslauer, et al. (1986). PNAS. 83, 3746-3750.

    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.

    Manuals

    The Product Manual 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 these document files: manual[Catalog Number].
    1. What has changed?
    2. Have the formulations or any other characteristics of these products changed now that they are manufactured by NEB?
    3. Can I continue to use the same reaction conditions if I use Phusion® products with the new product numbers?
    4. Are the prices and sizes of these products the same now that they are manufactured by NEB?
    5. Are the DNA fragments produced by Phusion® High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields?
    6. My template is GC-rich or supercoiled. How can I optimize my product yield using Phusion® High-Fidelity DNA Polymerase?
    7. What are the advantages to using Phusion® High-Fidelity DNA Polymerase?
    8. What is the error rate of Phusion® High-Fidelity DNA Polymerase?
    9. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® High-Fidelity DNA Polymerase?
    10. Why do I see no product or low yield on an agarose gel after a PCR using Phusion® High-Fidelity DNA Polymerase?
    11. Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion® High-Fidelity DNA Polymerase?
    12. Will Phusion® High-Fidelity DNA Polymerase incorporate dUTPs?
    13. Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion® High-Fidelity DNA Polymerase?
    14. I'd like to clone a fragment amplified with Phusion® High-Fidelity DNA Polymerase. Do I have to blunt-end clone?
    15. What should my primer concentration be when using Phusion® High-Fidelity DNA Polymerase/Phusion® High-Fidelity PCR Master Mix?
    1. PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit
    2. Protocol for a Routine PCR with Phusion® High-Fidelity PCR Kit
    3. PCR Optimization with Phusion® High-Fidelity PCR Kit

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