Klenow Fragment (2500 U)

A mesophilic DNA polymerase offered by QIAGEN, this enzyme is derived from the E. coli Polymerase I and exhibits DNA synthesis together with proofreading (3′→5′) nuclease activity. In the absence of the holoenzyme's (5′→3′) nuclease domain, the protein displays a moderate strand displacement activity during DNA synthesis. The enzyme is expressed as a truncated product of the E. coli PolA gene, produced by a recombinant E. coli strain carrying the Klenow Fragment gene, with a molecular weight of 68.2 KDa.

One unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 37°C. Specifications include >99% purity assessed by SDS-PAGE with silver-stain detection, single-stranded exonuclease activity functional at 50 U, double-stranded exonuclease activity functional at 50 U, double-stranded endonuclease activity showing no conversion at 50 U, and E. coli DNA contamination of <10 copies at 50 U as evaluated by TaqMan qPCR against the 16S rRNA locus. Specific activity is 5,000 U/mg.

The enzyme is supplied in 100 mM KPO4, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol (pH 7.5 at 25°C) and is accompanied by a 10X reaction buffer containing 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, and 10 mM DTT (pH 7.9 at 25°C). Recommended storage is −25°C to −15°C. Typical applications include removal of 3′ overhangs and filling-in of 5′ overhangs: dissolve DNA in 1X reaction buffer, add dNTPs to a final concentration of 33 µM each, add 1 U of polymerase per microgram of DNA, incubate at 25°C for 15 minutes, and stop the reaction by adding EDTA to a final concentration of 10 mM followed by heating at 75°C for 20 minutes. The product is intended for in vitro use only.