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  • T4 DNA Polymerase

    Description

    T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. This enzyme has a 3´→ 5´ exonuclease activity which is much more active than that found in DNA Polymerase I (E. coli).  Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5´→ 3´ exonuclease function.



    Highlights

    • Extreme fidelity (6)
    • Gap filling (no strand displacement activity)
    • Best enzyme for creating blunt ends
    • Isolated from a recombinant source

    Product Source

    Purified from a strain of E. coli that carries the T4 DNA Polymerase gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X
    BSA-2010 mg/ml

    Advantages and Features

    Applications

    • 3´ overhang removal to form blunt ends (1,2).
    • 5´ overhang fill-in to form blunt ends (1,2).
    • Single strand deletion subcloning (3).
    • Second strand synthesis in site-directed mutagenesis (4).
    • Probe labeling using replacement synthesis (1,2).

    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 37°C (5).

    Reaction Conditions

    1X NEBuffer 2
    Supplement with 10 mg/ml BSA
    Incubate at 12°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    100 mM KPO4
    1 mM DTT
    50% Glycerol
    pH 6.5 @ 25°C

    Heat Inactivation

    75°C for 20 min

    Molecular Weight

    Theoretical: 112000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    No

    Unit Assay Conditions

    1X NEBuffer 2, 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA and 50 µg/ml BSA.

    Error Rate

    ~ 1x10-6bases

    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. Incubate at temperature suggested for specific protocol. Refer to specific protocol to determine recommended dNTP concentrations.
    2. T4 DNA Polymerase is active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs and BSA.
    3. Protocol for blunting ends by 3´ overhang removal and 3´ recessed end fill-in:
      DNA should be dissolved in any 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes (1,2). CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.
    4. Add EDTA to a final concentration of 10 mM and heat inactivate at 75°C for 20 minutes.

    References

    1. Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. In F. M. Ausebel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl(Ed.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc.
    2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
    3. Dale, R., McClure, B. and Houchins, J. (1985). Plasmid. 13, 31-40.
    4. Kunkel, T.A., Roberts, J.D. and Zakour, R.A. (1987). Methods Enzymol.. 154, 367-382.
    5. Panet, A., van de Sande, J.H., Loewen, P.C. and Khorana, H.G. (1973). Biochemistry. 12, 5045-5050.
    6. Kunkel, T.A., Loeb, L.A. and Goodman, M.F. (1984). J. Biol. Chem.. 259, 1539-1545.

    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 T4 DNA Polymerase be used in other NEBuffers?
    2. Can T4 DNA Polymerase be used to blunt DNA?
    3. Can T4 DNA Polymerase be used to fill in 3' overhangs?
    4. Can T4 DNA Polymerase be used to remove 5' overhangs?
    5. Can T4 DNA Polymerase be heat inactivated?
    6. Are the nucleotides needed to remove a 3' overhang using T4 DNA Polymerase?
    7. What are the main causes of blunting reaction failure using T4 DNA Polymerase?
    8. Are NEB DNA Polymerases supplied with dNTPs?
    9. Can T4 DNA Polymerase be used in labeling reactions and partial fill in reactions?
    10. Is T4 DNA Polymerase active at room temperature?
    11. Is T4 DNA Polymerase the enzyme of choice for removing 3' overhangs and filling in 5' overhangs (3' recessed ends)?
    1. Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using T4 DNA Polymerase (M0203)

    Selection Tools

    Excess enzyme, temperatures above 12°C, or limited dNTP concentrations can cause excessive 3’ → 5’ exonuclease degradation, which eliminates blunt end formation.