T4 DNA Ligase (150000 U)

A recombinant ligase from QIAGEN, produced by an E. coli strain carrying the cloned bacteriophage T4 DNA ligase gene, with a molecular weight of 55,292 Daltons. The enzyme catalyzes the formation of a phosphodiester bond between a terminal 5′ phosphate and a 3′ hydroxyl group of duplex DNA or RNA, efficiently joining blunt and cohesive ends and repairing single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids. It is also effective for joining blunt or cohesive ends in double-stranded RNA when held together by a complementary DNA or RNA splint strand, and cannot join single-stranded nucleic acids. The enzyme is ATP-dependent.

The ligase is supplied at a concentration of 120,000 U/mL in 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol, pH 7.5 at 25°C, and is provided with a 10X T4 DNA Ligation Buffer composed of 500 mM Tris-HCl, 100 mM MgCl2, 50 mM DTT and 10 mM ATP, pH 7.6 at 25°C. Storage temperature is –25°C to –15°C. Quality control specifications include >99% purity, a specific activity of 300,000 U/mg, <1.0% released single-stranded exonuclease activity, <1.0% released double-stranded exonuclease activity, no conversion in double-stranded endonuclease testing, and <10 copies of E. coli DNA contamination. One unit is defined as the amount of enzyme required to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µL of 1X T4 DNA Ligase Buffer following a 30 minute incubation at 23°C.

For standard ligation reactions, components are combined and incubated at 25°C for 30 minutes, after which the DNA is purified by PCR clean-up column or diluted prior to transformation of 0.1–10 ng of ligation product. Single-insert ligations are optimal with an insert-to-vector ratio between 2:1 and 6:1; ratios above 6:1 promote the insertion of multiple fragments, while ratios below 2:1 reduce ligation efficiency. The product is developed, manufactured and sold for in vitro use only and is not suitable for administration to humans or animals.