DNA POLYMERASE I, KLENOW FRAGMENT FROM E

Description_x000D_ General description_x000D_ 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._x000D_ Application_x000D_ Suitable for:_x000D_ • DNA sequencing by the Sanger dideoxy method_x000D_ • Synthesis of the complementary strand of cDNA_x000D_ • Filling in 5′-overhangs in double stranded DNA to form blunt ends_x000D_ • Mutagenesis of DNA with second strand synthesis using oligonucleotides_x000D_ • Labeling DNA by the random primer method_x000D_ Components_x000D_ 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) ._x000D_ Unit Definition_x000D_ One unit converts 10 nanomoles of deoxyribonucleoside triphosphates into acid insoluble material in 30 min. at 37 °C._x000D_ Reconstitution_x000D_ 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._x000D_ Analysis Note_x000D_ 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.