DNA Polymerase Alpha (Human)

Unit definition: One unit is the amount of enzyme required to incorporate 1 nanomole of total nucleotide into acid-insoluble form in 60 min. at 37°C.

Storage conditions: Store at -80°C for long-term, or -20°C for short-term storage.

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, o.1 mM spermine, 0.05 mM each dCTP, dGTP, dTTP, dATP, (pH 7.0), [α-³H]dATP, and 20 µg activated calf thymus DNA. Incubation is at 37°C for 30 min. in a reaction volume of 50 µl.

Quality Control: The polymerase is tested for significant polymerase and primase activity. All preparations are assayed for contaminating endonuclease, 3’- and 5’-exonuclease, nonspecific RNase, and single- and double-stranded DNase activities.

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.

References:

1. Podust, V.N., Lavrik, O.I., Nasheuer, H.-P., and Grosse, F. (1989) FEBS Letters 245, 14-16