| 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. |
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| Catalog # | D3941 |
| 10X Reaction Buffer (D3941A) | 500mM Tris-HCl, pH 8.0 (25°C), 50mM magnesium chloride, 10mM DTT. |
| Unit Definition | 1u 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 |
| Applications | Suitable 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 Assay | 67mM 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 Control | Endodeoxyribonuclease 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 Assay | 0% 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 Assay | No 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 Assay | Klenow Fragment, exo-, was tested for DNA labeling by the random primer method. |
| Storage and Stability | For 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. |
| Source | E. coli |
| Concentration | ~5u/ul |
| Form | Supplied as a liquid in 25mM Tris-HCl, pH 7.4, 0.1mM EDTA, 1mM DTT, 50% glycerol. |
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| Important Note | This 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 names | EC=2.7.7.7; E. coli Supplied with Reaction Buffer |
