Practice question

Alkaline phosphatase dephosphorylates the 5' ends of cleaved vector DNA, removing the phosphate groups essential for phosphodiester bond formation by DNA ligase. Self-ligation requires both 5' phosphate and 3' hydroxyl ends; dephosphorylation prevents recircularization of empty vector without insert. This forces ligation to depend on phosphate contributed by foreign DNA insert, greatly increasing recombinant yield and reducing background colonies. Calf intestinal phosphatase and shrimp alkaline phosphatase are commonly used, followed by heat inactivation or column purification before ligation and bacterial transformation steps.