T4 DNA Polymerase Source: bacteriophage T4 of Escherichia coliDescription:• 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 37oC.Storage Conditions: Store at –20oC.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 22oC), 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 37oC 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.