E. coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β-NAD between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate cohesive termini. Single-stranded nucleic acids are not substrates for this enzyme.

Applications: Second-strand cDNA synthesis (1). T4 DNA Ligase alternative when blunt-end ligation is not required (2).

Source: Purified from E. coli 594 (Su- ) bearing λ lysogen gt4lop-11 lig+ S7 (3).

Performance and Quality Testing: Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.

Unit Definition: One unit is the amount of enzyme required to give 50% ligation of Hind III-digested λ DNA in 30 min. at 16°C in a final volume of 20 µl containing a 5´ termini concentration of 0.12 ´M (300 µg/ml).

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 in 20 µ