Unit definition: One unit is the amount of enzyme required to incorporate 1 nmol of total nucleotide into acid-insoluble form in 60 min. at 37°C.
Assay conditions: 60 mM Tris-HCl, pH 8.0, 5.0 mM magnesium acetate, 0.3 mg/ml bovine serum albumin, 1.0 mM dithiothreitol, 0.1 mM spermine, 0.05 mM each dCTP, dGTP, dTTP, dATP, (pH 7.0), [α-³H]dATP, and 10 µg activated calf thymus DNA. Incubation is at 37°C for 30 min. in a reaction volume of 50 µl.
Quality Control: All preparations are assayed for contaminating endonuclease, 3’- and 5’-exonuclease, nonspecific RNase, and single- and double-stranded DNase activities. The polymerase is also tested for significant primase activity.
Storage Buffer: 20 mM Tris-HCl, pH 8.0, 0.25 mM EDTA, 50 mM NaCl, 1 mM ß-mercaptoethanol, 0.1% Triton X-100, and 50% (v/v) glycerol.
Storage conditions: Store at -80°C for long-term, or -20°C for short-term storage.
1. Weinbeerg, D., personal communication.
2. Nasheuer, H., and Grosse, F. (1987) Biochemistry 26, 8458-8466.