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D8276

Sigma-Aldrich

DNA Polymerase I, Klenow Fragment from Escherichia coli

buffered aqueous glycerol solution

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CAS Number:
MDL number:
NACRES:
NA.53

grade

for molecular biology

Quality Level

form

buffered aqueous glycerol solution

mol wt

103 kDa

concentration

~3,000 units/mL

UniProt accession no.

foreign activity

Endonuclease, none detected

shipped in

wet ice

storage temp.

−20°C

Gene Information

Escherichia coli K12 ... polA(948356)

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This Item
D9380D1806D0690
form

buffered aqueous glycerol solution

form

buffered aqueous glycerol solution

form

liquid

form

aqueous glycerol solution

mol wt

103 kDa

mol wt

109 kDa

mol wt

94 kDa

mol wt

~374 kDa

concentration

~3,000 units/mL

concentration

5,000-15,000 units/mL

concentration

5 units/μL

concentration

≥2 unit/μL

UniProt accession no.

P00582

UniProt accession no.

P00582

UniProt accession no.

-

UniProt accession no.

P0AES4, P0AES6

foreign activity

Endonuclease, none detected

foreign activity

Endonuclease, none detected

foreign activity

-

foreign activity

-

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.

Pictograms

Health hazard

Signal Word

Danger

Hazard Statements

Precautionary Statements

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

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R B Wallace et al.
Science (New York, N.Y.), 209(4463), 1396-1400 (1980-09-19)
Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA
DNA polymerase versus DNA binding to the anticancer drug, cis-platin.
Bose, R.N., et al.
Inorgorganica Chimica Acta, 300, 937-937 (2000)
Sambrook, J.F., et al.
Molecular Cloning: A Laboratory Manual, 5-5 (1989)
H Klenow et al.
Proceedings of the National Academy of Sciences of the United States of America, 65(1), 168-175 (1970-01-01)
Purification of DNA polymerase from E. coli B has in two cases each time led to the isolation of two separate polymerase activities, enzyme A and enzyme B. Enzyme A was in contrast to enzyme B almost completely devoid of
L M Houdebine
Nucleic acids research, 3(3), 615-630 (1976-03-01)
E.Coli DNA polymerase I (Klenow subfragment) was used for the synthesis of complementary DNA with the mRNAs for rabbit milk proteins as templates. The cDNA formed, contained 200 nucleotides and represented about 20% of the mRNA template. The cDNA was

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