DNA Polymerase Alpha (Calf Thymus)

DNA polymerase alpha is a multi-subunit enzyme complex, which exhibits DNA polymerase and primase activities. It is isolated from calf thymus glands by immunoaffinity chromatography (1). This polymerase is used in DNA replication experiments.

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.

References:

1. Weinbeerg, D., personal communication.

2. Nasheuer, H., and Grosse, F. (1987) Biochemistry 26, 8458-8466.