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  • Phusion® Hot Start Flex 2X Master Mix

    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 applications. 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 that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase (1), 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.

    Phusion Hot Start Flex 2X Master Mix offers robust high fidelity performance and room temperature reaction setup in a convenient master mix format. The addition of an aptamer-based inhibitor allows room temperature reaction setup. 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. Reactions can also be optimized using the provided DMSO solution.

    Product Source

    An E. coli strain that carries the Phusion DNA Polymerase gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    DMSO100%

    Advantages and Features

    Applications

    • High-specificity PCR
    • Cloning
    • Long or Difficult Amplification
    • High-Throughput PCR

    Properties and Usage

    Storage Temperature

    -20°C

    Heat Inactivation

    No

    Unit Assay Conditions

    25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 200 μM dNTPs including [3H]- dTTP and 400 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.
    • 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, Human Genomic DNA, Master Mix)   :

      The polymerase master mix 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.

    Notes

    1. Phusion Hot Start Flex DNA Polymerase is unlike other enzymes and care must be taken when designing cycling protocols. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.
    2. Product specifications for individual components in the Phusion Hot Start Flex 2X Master Mix are available separately.

    References

    1. Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
    2. Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-290.

    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. Are the DNA fragments products by Phusion® Hot Start Flex 2X Master Mix blunt-ended or do they have the single-base 3’ overhang that Taq DNA Polymerase yields?
    2. What are the advantages of using Phusion® Hot Start Flex 2X Master Mix?
    3. Is the Phusion® Hot Start Flex 2X Master Mix a substitute for the discontinued Phusion Flash High-Fidelity PCR Master Mix (F-548S/L)?
    4. Why do I see no product or low yield on an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?
    5. Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?
    6. Will Phusion® Hot Start Flex 2X Master Mix incorporate dUTPs?
    7. Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?
    8. I'd like to clone a fragment amplified with Phusion® Hot Start Flex 2X Master Mix. Do I have to blunt-end clone?
    9. Does Phusion® Hot Start Flex 2X Master Mix exhibit a strand displacement activity?
    10. What should my primer concentration be when using Phusion® Hot Start Flex 2X Master Mix?
    11. How do I activate Phusion® Hot Start Flex DNA Polymerase?
    12. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® Hot Start Flex 2X Master Mix?
    1. Protocol for Phusion® Hot Start Flex 2X Master Mix

    Selection Tools

    Feature Articles

    Troubleshooting Guides

    Interactive Tools

    Phusion Hot Start Flex has different annealing temperature requirements than most PCR enzymes. Please check out www.neb.com/tmcalculator to help you determine your optimal Phusion annealing temperature.