Figure 13.4 : Mechanism of DNA ligase.

3. Alkalike phosphatase- Digested linear plasmid containing cohesive ends on both sites with phosphate has a tendency to recircularize (Figure 13.5). Removing terminal phosphate group prevents this possibility and for this purpose, alkaline phosphatase is used. Alkalike phosphatase removes 5'-terminal phosphate groups and in this condition, only in the presence of insert DNA as it will supply phosphate group at both ends to facilitate the ligation reaction (Figure 13.5).