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D8276 Sigma

DNA Polymerase I, Klenow Fragment from Escherichia coli

buffered aqueous glycerol solution

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Properties

Related Categories Application Index, Biochemicals and Reagents, Enzymes, Inhibitors, and Substrates, Modifying Enzymes, Molecular Biology,
grade   for molecular biology
form   buffered aqueous glycerol solution
mol wt   mol wt 103 kDa
concentration   ~3,000 units/mL
foreign activity   Endonuclease, none detected
shipped in   wet ice
storage temp.   −20°C
Gene Information   Escherichia coli K12 ... polA(948356)

Description

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) .

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.

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.

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.

Unit Definition

One unit converts 10 nanomoles of deoxyribonucleoside triphosphates into acid insoluble material in 30 min. at 37 °C.

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

Price and Availability


Biomedical Applications
Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
2

Documents

Certificate of Analysis

Protocols & Articles
Peer-Reviewed Papers
15

References

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