Merck, D8276-50UN, DNA Polymerase I, Klenow Fragment from Escherichia coli

General description
DNA polymerase I yields two fragments (small and large) upon protease digestion. The large fragment (Klenow fragment) loses the 5′ exonuclease activity that is present in the intact holoenzyme. However, it retains both the polymerase 5′→3′ activity and the 3′→5′ exonuclease activity of the native enzyme.
Application
Suitable for:
DNA sequencing by the Sanger dideoxy method
Synthesis of the complementary strand of cDNA
Filling in 5′-overhangs in double stranded DNA to form blunt ends
Mutagenesis of DNA with second strand synthesis using oligonucleotides
Labeling DNA by the random primer method

Preparation Note
The enzyme solution may be diluted with 50 mM Tris-HCl, pH 7.5, 100 mM ammonium sulfate, 10 mM 2-mercaptoethanol, and 1 mg/ml bovine serum albumin.
Analysis Note
The activity is assayed in a reaction mixture containing 50 mM potassium phosphate (pH 7.5), 3 mM MgCl₂, 1 mM 2-mercaptoethanol, 32.5 μM ³²P-dATP, 32.5 μM dTTP, 62.5 μg/ml poly(dA-dT) and 0.01-1 unit enzyme.
Other Notes
DNA Polymerase I is supplied as a solution in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM dithiothretol, and 50% glycerol (v/v) .
One unit converts 10 nanomoles of deoxyribonucleoside triphosphates into acid insoluble material in 30 min. at 37 °C.