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  • phi29 DNA Polymerase

    Description

    phi29 DNA Polymerase is the replicative polymerase from the Bacillus subtilis phage phi29 (Φ29) (1). This polymerase has exceptional strand displacement and processive synthesis properties (2). The polymerase has an inherent 3´→5' proofreading exonuclease activity (3).

    Highlights

    • Extreme processivity
    • Extreme strand displacement
    • High-Fidelity
    • Isolated from a recombinant source

    Product Source

    An E. coli strain that carries the phi29 DNA Polymerase gene from bacteriophage phi29

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    phi29 DNA Polymerase Reaction Buffer-2010X
    BSA-2010 mg/ml

    Advantages and Features

    Applications

    • Replication requiring a high degree of strand displacement and/or processive synthesis
    • High fidelity replication at moderate temperatures

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 0.5 pmol of dNTP into acid insoluble material in 10 minutes at 30°C. 

    Reaction Conditions

    1X phi29 DNA Polymerase Reaction Buffer
    Supplement with BSA
    Incubate at 30°C

    1X phi29 DNA Polymerase Reaction Buffer:
    50 mM Tris-HCl
    10 mM MgCl2
    10 mM (NH4)2SO4
    4 mM DTT
    pH 7.5 @ 25°C

    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

    65°C for 10 min

    Molecular Weight

    Theoretical: 67000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    +

    Unit Assay Conditions

    1X phi29 DNA Polymerase Reaction Buffer, 0.1 mg/ml BSA, 0.01 mg/ml HindIII-digested λ DNA, 0.2 µM dTTP including [3H]-dTTP, 0.2 mM dGTP, 0.2 mM dATP and 0.2 mM dCTP.

    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.

    Notes

    1. The presence of active reducing reagent in the reaction buffer is critical for this enzyme. While the reaction buffer supplied with the enzyme contains DTT, older buffer stocks or stocks that have been repeatedly frozen and thawed should be supplemented with 4 mM DTT to obtain maximal activity.
    2. If stock solutions of lesser concentration are needed, use Diluent F (NEB #B8006).

    References

    1. Blanco, L. and Salas, M. (1984). Proc. Natl. Acad. Sci. USA. 81, 5325-5329.
    2. Blanco. L., et al. (1989). J. Biol. Chem.. 264, 8935-8940.
    3. Garmendia, C., et al. (1992). J. Biol. Chem.. 267, 2594-2599.

    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. Can phi29 DNA Polymerase be used in other NEBuffers?
    2. Can phi29 DNA Polymerase be used to blunt DNA?
    3. Can phi29 DNA Polymerase be used to fill in 3' overhangs?
    4. Can phi29 DNA Polymerase be used to remove 5' overhangs?
    5. Are NEB DNA Polymerases supplied with dNTPs?
    6. Can phi29 DNA Polymerase be heat inactivated?
    7. At what rate does phi29 DNA Polymerase add nucleotides to a primed single-stranded template?

    Selection Tools

    DTT in the reaction buffer can oxidize quickly with >-20°C storage temperatures, extended storage, or multiple freeze/thaws, which can reduce amplification efficiency of phi29 DNA Polymerase.  Older buffer stocks or stocks that have been repeatedly frozen and thawed should be supplemented with 4 mM DTT to obtain maximal activity.