DNA Polymerase I Large (Klenow) Fragment

DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E. coli DNA Polymerase I that lacks the 5´→3´ exonuclease activity of intact DNA polymerase I but retains its 5´→3´ polymerase, 3´→5´ exonuclease and strand displacement activities. The 5´→3´ polymerase activity of Klenow Fragment can be used to fill in 5´-protruding ends with unlabeled or labeled dNTPs, to sequence single- or double-stranded DNA templates, for in vitro mutagenesis using synthetic oligonucleotides, for cDNA second-strand synthesis and to generate single-stranded DNA probes. The 3´→5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. The DNA-dependent DNA polymerase is provided with 10X Reaction Buffer [500mM Tris-HCl (pH 7.2 at 25°C), 100mM MgSO₄, 1mM DTT].

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References

1. Anderson, S. et al. (1980) Nucl. Acids Res. 8, 1731–43.
2. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–7.
3. Wallace, R.B. et al. (1980) Science 209, 1396–400.
4. Houdebine, L.M. (1976) Nucl. Acids Res. 3, 615–30.
5. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6–13.
6. Tabor, S. and Struhl, K. (1987) In: Current Protocols in Molecular Biology, Ausubel, F.M. et al., eds., John Wiley and Sons, New York, NY.
7. Joyce, C.M. and Grindley, N.D.F. (1983) Proc. Natl. Acad. Sci. USA 80, 1830–4.

Storage Buffer: 50mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerol.

Source: Recombinant E. coli strain.

QC Tests: Activity, SDS-PAGE/purity, endonuclease.

Unit Definition: One unit is defined as the amount of enzyme that incorporates 10nmol of total deoxyribonucleotides into TCA-insoluble material in 30 minutes at 37°C. The reaction conditions are: 67mM potassium phosphate (pH 7.5 at 25°C), 6.7mM MgCl₂, 1mM DTT, 50μg/ml activated calf thymus DNA and 33μM each of dATP, dCTP, dGTP and dTTP (a mix of unlabeled and [³H]dTTP).