DNA Polymerase Beta (Human)
Description:
- simplest DNA polymerase known in both size and catalysis (1).
- a repair polymerase able to synthesize DNA beyond the end of gap or nick with simultaneous displacement of the non-replicated strand (2).
- fills gaps or nicks (3).
Unit definition: One unit is the amount of enzyme required to incorporate 1 nmole of total nucleotide into acid-insoluble form in 60 min. at 37°C.
1 x Reaction Buffer: 50 mM Tris-HCl (pH 8.7), 10 mM MgCl2, 0.4 mg/ml bovine serum albumin, 1.0 mM dithiothreitol, 100 mM KCl, 15% glycerol.
Storage Buffer: 20 mM Tris-HCl, pH 8.0, 1.0 mM dithiothreitol, 0.1 mM EDTA, 0.2 M NaCl and 50% (v/v) glycerol.
Assay conditions: 50 mM Tris-HCl, pH 8.7, 10 mM MgCl2, 0.4 mg/ml bovine serum albumin, 1.0 mM dithiothreitol, 100 mM KCl, 15% glycerol, 0.05 mM each dCTP, dGTP, dTTP, [α-32P]dATP and 10 µg activated DNA. Incubation is at 37°C for 15 min in a reaction volume of 50 µl.
Quality Control: All preparations are assayed for contaminating endonuclease and exonuclease activities.
Storage conditions: Store at -20°C.
References:
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Abbotts, J., SenGupta, D.N., Zmudzka, B., Widen, S.G., Notario, V., and Wilson, S.H. (1988) Biochemistry 27, No. 3, 901-909.
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Nowak, R., Kulik, J., and Siedlecki, J.A. (1987) Acta Biochim. Pol. 34, 205-215.
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Wang, T.S-F., and Korn, D., (1980) Biochemistry 19, 1782-1790.
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.
1 x Reaction Buffer: 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.
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.
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.
References:
1. Podust, V.N., Lavrik, O.I., Nasheuer, H.-P., and Grosse, F. (1989) FEBS Letters 245, 14-16