Ligation of DNA is a critical step in many modern molecular biology workflows. The sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks are enzymatically catalyzed by DNA ligases. The formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate of adjacent DNA residues proceeds in three steps: Initially, the ligase is self-adenylated by reaction with free ATP. Next, the adenyl group is transferred to the 5'-phosphorylated end of the "donor" strand. Lastly, the formation of the phosphodiester bond proceeds after reaction of the adenylated donor end with the adjacent 3' hydroxyl acceptor and the release of AMP. In living organisms, DNA ligases are essential enzymes with critical roles in DNA replication and repair. In the lab, DNA ligation is performed for both cloning and non-cloning applications.