Description

• 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 methodSynthesis of the complementary strand of cDNAFilling in 5′-overhangs in double stranded DNA to form blunt endsMutagenesis of DNA with second strand synthesis using oligonucleotidesLabeling DNA by the random primer method
• Components: 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) .
• Unit Definition: One unit converts 10 nanomoles of deoxyribonucleoside triphosphates into acid insoluble material in 30 min. at 37 °C.
• Reconstitution: 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.