Exonuclease I (30000 U)

A 3′→5′ exonuclease from QIAGEN, this enzyme selectively digests primers and single-stranded DNA while leaving double-stranded DNA intact. It is PCR-compatible and thermolabile, and can be heat inactivated by incubation at 80°C for 15 minutes. The enzyme preferentially degrades single-stranded oligonucleotide primers in reactions containing amplification products or other double-stranded DNA sources. Digestion is inhibited where the terminal 3'-OH group is blocked by phosphoryl or acetyl groups.

The enzyme works well in most molecular biology buffers containing magnesium over 1.5 mM. It is supplied in 10 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.5 mM EDTA and 50% glycerol at pH 7.5 (25°C), with a recommended storage temperature of –25°C to –15°C. The enzyme is purified from a strain of E. coli expressing the recombinant Exonuclease I gene and has a molecular weight of 54.5 kDa.

One unit is defined as the amount of enzyme required to produce 10 nmol of acid-soluble total nucleotide in 30 minutes at 37°C. Quality specifications include a specific activity of 185,000 U/mg, physical purity >99% by SDS-PAGE, no detectable double-stranded endonuclease activity (200 U tested, no conversion), and <10 copies of E. coli DNA contamination (200 U tested). A documented application is the removal of single-stranded DNA from PCR products in a one-tube cleanup.