85-1343-62　DNA Polymerase I Klenow Fragment, Exo-Minus, Recombinant 1500U　D3941

特徴

• 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.
• 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:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt 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.