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  • DNA Polymerase I (E. coli)

    Description

    DNA Polymerase I (E coli) is a DNA-dependent DNA polymerase with inherent 3´→ 5´ and 5´→ 3´ exonuclease activities (1). The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation.


    Highlights

    • Nick translation of DNA
    • Second strand cDNA synthesis
    • Supplied with 10X Reaction Buffer

    Product Source

    An E. coli strain that carries an overexpressed copy of the polA gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Advantages and Features

    Applications

    • Nick translation of DNA to obtain probes with a high specific activity (2)
    • Second strand synthesis of cDNA (3,4)

    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.

    Reaction Conditions

    1X NEBuffer 2
    Incubate at 37°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

    25 mM Tris-HCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    75°C for 20 min

    Molecular Weight

    Theoretical: 103000 daltons

    5' - 3' Exonuclease

    Yes

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    No

    Unit Assay Conditions

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

    Error Rate

    < 9x10-6bases

    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. DNase I is not included with this enzyme and must be added for nick translation reactions.
    2. DNA Polymerase I (E.coli) is active in all four NEBuffers when supplemented with dNTPs (not included).

    References

    1. Lehman, I.R. (1981). In P.D. Boyer(Ed.), The Enzymes. 14A, 16-38. San Diego: Academic Press.
    2. Meinkoth, J. and Wahl, G.M (1987). S.L. Berger and A.R. Kimmel(Ed.), Methods Enzymol.. 152, 91-94. San Diego: Academic Press.
    3. Gubler, U. and Hoffmann, B.J. (1983). Gene. 25, 263-269.
    4. D'Alessio, J.M. and Gerard, G.F. (1988). Nucl. Acids Res.. 16, 1999-2014.
    5. 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 DNA Polymerase I (E. coli) be used in other NEBuffers?
    2. Can DNA Polymerase I (E. coli) be used to blunt DNA?
    3. Can DNA Polymerase I (E. coli) be used to fill in 3' overhangs?
    4. Why are there artifacts in my blots using the nick translated probe?
    5. Are NEB DNA Polymerases supplied with dNTPs?
    6. Can DNA Polymerase I (E. coli) be used to remove 5' overhangs?
    7. Can DNA Polymerase I (E. coli) be heat inactivated?
    8. Can DNA Polymerase I (E. coli) be used in nick translation protocols?
    9. Is nick translation the best way to make a labeled probe?

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