| 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 3terminus of a blunt-ended DNA substrate in a non-template directed fashion. 10X Reaction Buffer (D3941A): 500mM Tris-HCl, pH 8.0 (25C), 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 37C 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 37C. 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 37C. 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 37C. Functional Assay: Klenow Fragment, exo-, was tested for DNA labeling by the random primer method. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. 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. |
