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  • T7 DNA Polymerase (unmodified)

    Description

    T7 DNA Polymerase catalyzes the replication of T7 phage DNA during infection. The protein dimer has two catalytic activities: DNA polymerase activity and strong 3´→ 5´ exonuclease (1,2,3). The high fidelity and rapid extension rate of the enzyme make it particularly useful in copying long stretches of DNA template.

    Highlights

    • Isolated from a recombinant source
    • Second strand synthesis in site-directed mutagenesis
    • Supplied with 10X Reaction Buffer
    • Gap filling reaction (no strand displacement)

    Product Source

    T7 DNA Polymerase consists of two subunits: T7 gene 5 protein (84 kilodaltons) and E.coli thioredoxin (12 kilodaltons) (1,4-7). Each protein is cloned and overexpressed in a T7 expression system in E. coli (4).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T7 DNA Polymerase Reaction Buffer10X
    BSA-2010 mg/ml

    Advantages and Features

    Applications

    • Second strand synthesis in site-directed mutagenesis protocols (8).

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate10 nmoles of dNTP into acid insoluble material in 30 minutes at 37°C .

    Reaction Conditions

    1X T7 DNA Polymerase Reaction Buffer
    Supplement with BSA

    1X T7 DNA Polymerase Reaction Buffer:
    20 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM KPO4
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.0 @ 25°C

    Heat Inactivation

    75°C for 20 min

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    No

    Unit Assay Conditions

    100 mM KCl, 20 mM Tris-HCl (pH 7.6), 6 mM MgCl2, 0.5 mM Dithiothreitol, 0.1 mM EDTA, 50 μg/ml BSA, 150 μM dNTPs including [3H]-dTTP and 0.162 mg/ml activated calf thymus DNA.

    Error Rate

    ~ 15x10-6bases

    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. The high polymerization rate of the enzyme makes long incubations unnecessary.
    2. T7 DNA Polymerase is not suitable for DNA sequencing.

    References

    1. Hori, K. et al. (1979). J. Biol. Chem.. 254, 11598-11604.
    2. Engler, M.J. et al. (1983). J. Biol. Chem.. 258, 11165-11173.
    3. Nordstrom, B. et al. (1981). J. Biol. Chem.. 256, 3112-3117.
    4. Studier, F.W. et al. (1990). Goeddel, D.V.(Ed.), In Methods Enzymol.. 185, 60-89. San Diego: Academic Press.
    5. Grippo, P. and Richardson, C.C. (1971). J. Biol. Chem. 246, 6867-6873.
    6. Modrich, P. and Richardson, C.C. (1975). J. Biol. Chem. 250, 5515-5522.
    7. Adler, S. and Modrich, P. (1979). J. Biol. Chem. 254, 11605-11614.
    8. Bebenek, K. and Kunkel, T.A. (1989). Nucl. Acids Res.. 17, 5408.
    9. Mattila, P., et al. (1991). Nucleic Acids Res.. 19, 4967-4973.

    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 T7 DNA Polymerase be used in other NEBuffers?
    2. Can T7 DNA Polymerase be used to blunt DNA?
    3. Can T7 DNA Polymerase be used to fill in 3' overhangs?
    4. Can T7 DNA Polymerase be used to remove 5' overhangs?
    5. Can T7 DNA Polymerase be heat inactivated?
    6. Are NEB DNA Polymerases supplied with dNTPs?
    7. Are deoxynucleotides needed to remove a 3' overhang using T7 DNA Polymerase?
    8. When should native T7 DNA Polymerase be used?
    9. What is the oligonucleotide-directed mutagenesis without phenotypic selection method?
    10. Can the native T7 DNA Polymerase be used for sequencing?
    11. Does NEB carry modified T7 Polymerase?
    12. Can T7 DNA Polymerase be used in labeling reactions and partial fill in reactions?

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