Description
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 MgSO 4 , 1mM DTT].
References
Anderson, S. et al. (1980) Nucl. Acids Res. 8 , 1731–43.
Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74 , 5463–7.
Wallace, R.B. et al. (1980) Science 209 , 1396–400.
Houdebine, L.M. (1976) Nucl. Acids Res. 3 , 615–30.
Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132 , 6–13.
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.
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 MgCl 2 , 1mM DTT, 50μg/ml activated calf thymus DNA and 33μM each of dATP, dCTP, dGTP and dTTP (a mix of unlabeled and [ 3 H]dTTP).
