D8276

DNA Polymerase I, Klenow Fragment from Escherichia coli

buffered aqueous glycerol solution

• CAS Number: 9012-90-2
• MDL number: MFCD00283091
• UNSPSC Code: 12352204
• NACRES: NA.53

Properties

• grade: for molecular biology
• Quality Level: 200
• form: buffered aqueous glycerol solution
• mol wt: 103 kDa
• concentration: ~3,000 units/mL
• UniProt accession no.: P00582
• foreign activity: Endonuclease, none detected
• shipped in: wet ice
• storage temp.: −20°C
• Gene Information: Escherichia coli K12 ... polA(948356)

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 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

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.

Safety Information

• Pictograms: GHS08
• Signal Word: Danger
• Hazard Statements: H334
• Precautionary Statements: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Storage Class Code: 10 - Combustible liquids
• WGK: WGK 1
• Flash Point(F): Not applicable
• Flash Point(C): Not applicable
• Personal Protective Equipment: dust mask type N95 (US), Eyeshields, Gloves