Klenow (3'-5' exo-) (HC) (10000 U)

A recombinant mesophilic DNA polymerase supplied by QIAGEN, this enzyme is the large fragment of E. coli DNA polymerase I rendered deficient in both proofreading (3'→5') and nick-translation (5'→3') exonuclease activities. It carries the D355A and E357A mutations and is expressed as a truncated product of the E. coli PolA gene in a recombinant E. coli strain. The enzyme displays moderate strand displacement activity during DNA synthesis and retains 5'→3' DNA polymerase activity.

The lack of both exonuclease activities makes it well suited to dA/dT tailing (A-tailing) and stable end-labeling of DNA. Its terminal transferase activity adds a single adenine or thymine nucleotide to the 3' ends of blunt-ended DNA fragments, enabling direct ligation of sequencing adapters during next-generation sequencing library preparation. The enzyme is also preferred for labeling DNA with modified nucleotides such as fluorescent bases because it cannot excise mismatched bases.

Specifications include purity greater than 99%, specific activity of 10,000 U/mg, and a molecular weight of 68,100 Daltons. The product is provided at a concentration of 50,000 U/mL and should be stored at -25°C to -15°C. 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. Quality control testing at 500 U shows <10% released in single-stranded exonuclease assays, <1% released in double-stranded exonuclease assays, and no conversion in double-stranded endonuclease assays. E. coli DNA contamination is below 10 copies and UDG activity is below 20 U/mL. The enzyme is supplied in 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol (pH 7.5 at 25°C), together with 10X Blue Buffer containing 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, and 10 mM DTT (pH 7.9 at 25°C). For in vitro use only.