T4 DNA Polymerase

Source: bacteriophage T4 of Escherichia coli

Description:

· Exhibits 5'->3' polymerase and 3'->5' exonuclease activities (1, 2).

· Adds labeled nucleotides to the recessed 3'-ends of DNA fragments.

· The polymerase requires the presence of a single-stranded DNA template and a primer.

· Exonuclease, stronger than that found in DNA Polymerase I, is more active on single-stranded DNA than on double-stranded DNA.

· Ultrapure recombinant enzyme.

· Exonuclease activity can be used to remove one or a few nucleotides from 3’-end of double–stranded DNA.

· Enzyme suitable for:

o 3’ overhang removal to form blunt ends (3,4)

o 5’ fill-in to form blunt ends (3,4)

o probe labeling using replacement synthesis (3,4)

o single strand deletion subcloning (5)

o 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^oC.

Storage Conditions: Store at –20^oC.

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^oC), 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 [a-³²P]dATP. Incubation is at 37^oC for 30 min in a reaction volume of 100 µl (1).

Quality Control: All preparations are tested for contaminating endonuclease activity.

References:

1. Goulian, M., Lucas, Z.J. and Kornberg, A. (1968) J. Biol. Chem. 243, 627-638.

2. Lehman, I.R. (1981) Enzymes 14, 51-65.

3. 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.

4. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, pp. 5.44-5.47, Cold Spring Harbour.

5. Dale, R., McClure, B. and Houchins, J., (1985) Plasmid 13, 31-40.

6. Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Methods Enzymol.154, 367-382.

T4 DNA Polymerase is the mezophilic polymerase, exhibiting very strong 3'->5' exonuclease activity.