DNA Polymerase I, Large (Klenow) Fragment is a product of E. coli DNA Polymerase I which lacks the N-terminal 324 amino acids. This enzyme lacks the 5’→ 3′ exonuclease activity of intact DNA Polymerase I, but does exhibit the 5’→ 3′ DNA polymerase and 3’→ 5′ exonuclease activities
The physical purity of this enzyme is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
E.coli cells carrying E. coli polA gene without its N-terminal exonuclease domain.
- 3´-overhangs removal to form blunt ends (1).
- 5´-overhangs fill-in to form blunt ends (1).
- DNA library preparation for Next-generation sequencing (2).
- 3′-end-labeling of DNA fragments using α-32P deoxynucleotides.
- Second strand cDNA synthesis.
- Single-stranded DNA probes generation.
- DNA Polymerase I, Large (Klenow) Fragment
- 10x Klenow Reaction Buffer
50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM β-mercaptoethanol, 1 mM DTT, 50% (v/v) glycerol.
10x Klenow 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, Klenow Fragment is free from detectable endonuclease and RNase activities.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
- Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.