E.coli DNA Polymerase I is a DNA-dependent DNA polymerase which has both 3´→ 5´ and 5´→ 3´ exonuclease activities (1). The 5´→ 3´ exonuclease removes nucleotides from the growing DNA chain and allows nick-translation.
The physical purity of this enzyme is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
E. coli strain carrying recombinant E. coli polA gene.
- Nick translation of DNA to generate highly specific DNA probes (2).
- Second strand cDNA synthesis (3).
- DNA Polymerase I
- 10x DNA Polymerase I Reaction Buffer
50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM β-mercaptoethanol, 1 mM DTT and 50% (v/v) glycerol.
10X Reaction Buffer
500 mM Tris-HCl, 100 mM MgCl2, 50 mM dithiothreitol (DTT), pH 7.5 @ 25 °C.
Inactivated by heating at 75 °C for 20 min.
Quality Control Assays
DNA Polymerase I is free from detectable endonuclease and RNase activities.
- Lehman, I. R (1981). In P.D. Boyer(Ed.), The Enzymes. 14A, 16-38. San Diego: Academic Press.
- Meinkoth, J and Wahl, G. M (1987). S. L. Berger and A. R. Kimmel (Ed.), Methods Enzymol.152, 91-94. San Diego: Academic Press.
- Gubler, U and Hoffmann, B. J (1983). Gene. 25, 263-269.