T3 DNA Ligase (900000 U)

From QIAGEN's assay technologies portfolio, this recombinant ligase is produced by an E. coli strain carrying the cloned T3 DNA Ligase gene and has a molecular weight of 39.4 KDa. The enzyme catalyzes the formation of a phosphodiester bond between a 5ʹ phosphate and a 3ʹ hydroxyl terminus in duplex DNA, joining blunt-end and cohesive-end termini and repairing single-stranded nicks in duplex DNA. It is more efficient at joining A- and T-overhangs than C- and G-matched ends, retains 95% of its activity in 1.0 M NaCl or KCl, and shows an optimal salt concentration of 300 mM.

At a concentration of 3,000,000 U/mL, the enzyme displays a specific activity of 3,000,000 U/mg with purity of >99% as assessed by SDS-PAGE. Quality testing at 30,000 units shows <1.0% released single-stranded exonuclease activity, <1.0% released double-stranded exonuclease activity, no detectable double-stranded endonuclease conversion, and <10 copies of E. coli DNA contamination by TaqMan qPCR. The enzyme is supplied in 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol (pH 7.5 at 25°C), and is shipped with a 2X Rapid Ligation Buffer containing 132 mM Tris-HCl, 20 mM MgCl2, 2 mM DTT, 2 mM ATP, and 15% PEG 6000 (pH 7.6 at 25°C).

One unit is defined as the amount of enzyme required to ligate 50% of 100 ng of DNA fragments with cohesive termini in 30 minutes at 23°C. Recommended storage is -25°C to -15°C, and the product is intended for in vitro use only.