E. coli DNA ligase catalyzes 3´ hydroxyl groups in the presence of β-NAD A phosphodiester bond is formed between the double-stranded DNA and the 5´ phosphate cohesive terminus. Single-stranded nucleic acids are not substrates for this enzyme.

Applications: Second strand cDNA synthesis (1). When blunt-end ligation is not required, choose T4 DNA ligase (2).

Source: from E. colilig⁺ S7 >594 (Su^-) was purified to obtain (3).

Performance and quality testing: Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; tested for ligation efficiency.

Unit Definition:One unit is defined in a final volume of 20 µl containing a 5´ end concentration of 0.12 µM (300 µg/ml) in 30 minutes at 16°C Amount of enzyme required to complete 50% ligation ofHind III-digested lambda DNA.

Unit reaction conditions:18.8 mM Tris-HCl (pH 8.3), 90.6 mM KCl, 4.6 mM MgCl₂, 3.8 mM DTT, 0.15 mM λ-NAD, 10 mM (NH₄)₂ SO₄, a total of 20 µl solution, react for 1 hour at 16°C.