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You are here:Home » Cloning » Cloning-Enzymes » DNA Polymerase I Klenow Fragment, Exo-Minus, Recombinant

DNA Polymerase I Klenow Fragment, Exo-Minus, Recombinant

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Specifications

Klenow fragment, exo, exhibits 5’-3’ polymerase activity of DNA polymerase I, E. coli. It lacks 3’-5’ and 5’-3’ exonuclease activities. It is purified from an E. coli strain carrying a modified DNA polymerase I large fragment overproducing plasmid. Klenow Fragment, exo-, is not recommended for fill-in reactions prior to DNA ligation. It frequently adds one or more extra nucleotides to the 3’terminus of a blunt-ended DNA substrate in a non-template directed fashion.
Catalog #D3941
10X Reaction Buffer (D3941A)500mM Tris-HCl, pH 8.0 (25°C), 50mM magnesium chloride, 10mM DTT.
Unit Definition1u incorporates 10nm of total deoxynucleotides into a DE-81 absorbable form using poly(dA-dT)•poly(dA-dT) as a template-primer in 30 minutes at 37ºC
ApplicationsSuitable for use in labeling of DNA by the random primer method; labeling recessed 3’-termini of double-stranded DNA; DNA sequencing by the method of Sanger, F., et al.; Strand Displacement Amplification (SDA).
Activity Assay67mM potassium phosphate, pH 7.4, 6.7mM magnesium chloride, 1mM 2-mercaptoethanol, 0.033mM dATP, 0.033mM dTTP, 0.4MBq/ml [3H]-dTTP, 62.5ug/ml poly(dA-dT)•poly(dA-dT).
Quality ControlEndodeoxyribonuclease Assay:
No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 20u of Klenow Fragment, exo-. with 1ug of pBR322 DNA in 50ul of reaction buffer for 4 hours at 37°C.
Exodeoxyribonuclease Assay0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 20u of Klenow Fragment, exo-, with 1ug of sonicated E. coli [3H]-DNA in 50ul of reaction buffer for 4 hours at 37°C.
Labeled Oligonucleotide AssayNo detectable degradation of single- stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of Klenow Fragment, exo-, for 4 hours at 37°C.
Functional AssayKlenow Fragment, exo-, was tested for DNA labeling by the random primer method.
Storage and StabilityFor long-term storage, store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
SourceE. coli
Concentration~5u/ul
FormSupplied as a liquid in 25mM Tris-HCl, pH 7.4, 0.1mM EDTA, 1mM DTT, 50% glycerol.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Alternate namesEC=2.7.7.7; E. coli Supplied with Reaction Buffer


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