Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase.

Hexamine cobalt chloride (HCC) increases the efficiency of blunt end ligation by T4 DNA ligase about 50 fold. Maximum stimulation occurs when standard buffers for ligation are supplemented with 1 mM HCC. All the ligation events are intermolecular regardless of the initial DNA concentration. In the presence of monovalent cations (eg. 25 mM KCl) HCC still increases the extent of T4 catalyzed ligation but intramolecular ligation products are also formed. Therefore, intermolecular ligation can be performed rapidly and at low DNA concentrations.