Abstract: Methods and kits for covalently joining a 3? nucleic acid fragment having a 5?-hydroxyl terminus to a 5? nucleic acid fragment having a 3?-phosphate terminus are disclosed. The methods include the step of contacting the 3?-phosphate terminus of a first nucleic acid molecule and the 5?-hydroxyl terminus of a second nucleic acid molecule with an isolated 2?,3?-cyclic phosphate RNA ligase (RtcB) and a purine triphosphate in the presence of manganese (II) ion, whereby the 3?-phosphate terminus of the first nucleic acid molecule and the 5?-hydroxyl terminus of the second nucleic acid molecule are covalently joined. Although the purine triphosphate used is generally GTP or dGTP, if the method is performed in the presence of an Archease, any purine triphosphate may be used. Accordingly, the disclosed kits include isolated RtcB, along with a purine triphosphate and/or an isolated Archease.