T7 DNA Ligase (900000 U)

A recombinant DNA ligase from QIAGEN, T7 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5ʹ phosphate and a 3ʹ hydroxyl terminus in duplex DNA. The enzyme efficiently joins cohesive end termini and repairs single-stranded nicks, but is not very effective at blunt-end ligation under normal reaction conditions. Adding high concentrations of PEG 6000 at ≥ 20% (w/v) can stimulate activity and yield measurable blunt-end results.

The protein is produced by a recombinant E. coli strain carrying the T7 DNA Ligase gene and is supplied in 20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C. The accompanying 2x Rapid Ligation Buffer contains 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 required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.

For sticky-end ligation, the recommended vector:insert DNA molar ratio is 1:3. Reactions are incubated at 25°C for 15–30 minutes in a total volume of 20 µL, with the ligase added last. Quality specifications include >99% purity and a specific activity of 3,000,000 U/mg, with <1% single-stranded exonuclease release, <1% double-stranded exonuclease release, no double-stranded endonuclease conversion, and <10 copies of E. coli DNA contamination.