T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA with blunt or cohesive end. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids but has no activity on single-strand nucleic acids. T4 DNA Ligase requires ATP as a cofactor.
One unit is the amount of enzyme required to give 50% ligation of Hind III fragments of λDNA (5´ DNA termini concentration of
0.12 μM, 200 μg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1×T4 DNA Ligase Buffer.