T4 DNA Ligase should be used to ligate cohesive ends (10 minutes at room temperature) or blunt ends (2 hours at room temperature) or if the ligation is to be done overnight. T4 DNA Ligase should be used if heat inactivation is required. Concentrated T4 DNA Ligase is suggested for ligating single base overhangs or for large constructs which require longer incubation times to circularize, such as BAC (Bacterial Artificial Chromosome) construction.