T4 DNA Polymerase
Source: bacteriophage T4 of Escherichia coli
T4 DNA Polymerase is the mezophilic polymerase, exhibiting very strong 3'->5' exonuclease activity.
Description:
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Exhibits 5'->3' polymerase and 3'->5' exonuclease activities (1, 2).
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Adds labeled nucleotides to the recessed 3'-ends of DNA fragments.
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The polymerase requires the presence of a single-stranded DNA template and a primer.
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Exonuclease, stronger than that found in DNA Polymerase I, is more active on single-stranded DNA than on double-stranded DNA.
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Ultrapure recombinant enzyme.
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Exonuclease activity can be used to remove one or a few nucleotides from 3’-end of double–stranded DNA.
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Enzyme suitable for:
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3’ overhang removal to form blunt ends (3,4)
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5’ fill-in to form blunt ends (3,4)
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probe labeling using replacement synthesis (3,4)
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single strand deletion subcloning (5)
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second strand synthesis in site-directed mutagenesis (6)
Unit Definition: One unit is the amount of enzyme that catalyzes the incorporation of 10 nmoles of total nucleotide into acid-insoluble product in 30 min at 37°C.
Storage Conditions: Store at –20°C.
Storage Buffer: 20 mM potassium phosphate (pH 6.5), 5 mM dithiothreitol and 50% (v/v) glycerol.
Assay Conditions: 67 mM Tris-HCl (pH 8.8 at 22°C), 6.7 mM MgCl2, 10 mM dithiothreitol, 16.6 mM ammonium sulfate, 6.7 µM EDTA, 20 µg bovine serum albumin, 45 µg activated calf thymus DNA and 0.033 mM each of dCTP, dGTP, dTTP and [ α-³²P]dATP. Incubation is at 37°C for 30 min in a reaction volume of 100 µl (1).
Quality Control: All preparations are tested for contaminating endonuclease activity.
References:
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Goulian, M., Lucas, Z.J. and Kornberg, A. (1968) J. Biol. Chem. 243, 627-638.
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Lehman, I.R. (1981) Enzymes 14, 51-65.
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Tabor, S. and Struhl, K (1989) in Current Protocols in Molecular Biology (Ausubel, F. M., et al., eds) pp. 3.5.10-3.5.12, John Wiley&Sons, New York.
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Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, pp. 5.44-5.47, Cold Spring Harbour.
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Dale, R., McClure, B. and Houchins, J., (1985) Plasmid 13, 31-40.
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Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Methods Enzymol.154, 367-382.