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  • Phusion® Hot Start Flex DNA Polymerase

    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 DNA Polymerase offers robust, high fidelity performance and room temperature reaction setup. The addition of an aptamer-based inhibitor allows room temperature reaction setup. Phusion Hot Start Flex DNA Polymerase is supplied with standard 5X Phusion HF Buffer, as well as 5X Phusion GC Buffer, which can be used for complex or GC-rich templates. Each of these buffers contains MgCl2 (1.5 mM at the final [1X] reaction concentration). Reactions can also be optimized using the provided DMSO or MgCl2 solutions.

    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.

    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
    Phusion® HF Buffer-205X
    Phusion® GC Buffer Pack-205X
    MgCl2 solution50 mM
    DMSO100%

    Advantages and Features

    Features

    • High fidelity & robustness
    • High specificity, room temperature reaction setup

    Applications

    • High-specificity PCR
    • Cloning
    • Long or Difficult Amplification
    • High-Throughput 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 74°C.

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    1X stabilizers
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    5' - 3' Exonuclease

    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.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • 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.
    • 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. Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
    2. Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-029.

    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. Is Phusion® Hot Start Flex DNA Polymerase the same as Phusion Hot Start DNA Polymerase (#F-540S/L) or Phusion Hot Start II DNA Polymerase (#F-549S/L), which were previously sold by NEB?
    2. Are the DNA fragments products by Phusion® Hot Start Flex DNA Polymerase blunt-ended or do they have the single-base 3’ overhang that Taq DNA Polymerase yields?
    3. What are the advantages of using Phusion® Hot Start Flex DNA Polymerase?
    4. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® Hot Start Flex DNA Polymerase?
    5. Why do I see no product or low yield on an agarose gel after a PCR using Phusion® Hot Start Flex DNA Polymerase?
    6. Will Phusion® Hot Start Flex DNA Polymerase incorporate dUTPs?
    7. Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion® Hot Start Flex DNA Polymerase?
    8. I'd like to clone a fragment amplified with Phusion® Hot Start Flex DNA Polymerase. Do I have to blunt-end clone?
    9. I see foaming when my Phusion® Hot Start Flex-amplified PCR products are spotted on microarray slides.
    10. What should my primer concentration be when using Phusion® Hot Start Flex DNA Polymerase?
    11. How does the fidelity of Phusion® DNA Polymerase compare to Taq DNA Polymerase?
    12. Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion Hot Start Flex DNA Polymerase?
    13. Does Phusion® Hot Start Flex DNA Polymerase exhibit a strand displacement activity?
    14. How do I activate Phusion® Hot Start Flex DNA Polymerase?
    1. Protocol for Phusion® Hot Start Flex DNA Polymerase

    Selection Tools

    Feature Articles

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Phusion Hot Start Flex has different annealing temperature requirements than most PCR enzymes.
    Please check out the NEB Tm Calculator to help you determine your optimal Phusion annealing temperature.