How can calf intestinal alkaline phosphatase (CIAP) be inactivated after a dephosphorylation reaction?
Calf intestinal alkaline phosphatase, or CIAP, is often used in subcloning experiments to remove phosphate groups from the 5 ends of DNA molecules. The enzyme can be difficult to inactivate completely so it is important not to add excess enzyme. Due to the high activity of the enzyme (0.01 unit is sufficient to dephosphorylate 1pmol of DNA ends), any carryover of CIAP into the ligation reaction can drastically reduce the efficiency of the reaction. There are several methods to inactivate the enzyme. First, CIAP can be digested by adding EDTA (5mM), SDS (0.5%) and proteinase K (50g/ml) (final concentrations). The digestion is allowed to proceed at 56C for 30 minutes at which time the proteinase K can be inactivated by heating at 70C for 10-15 minutes. Second, EDTA can be added to a final concentration of 5mM and the reaction heated at 75C for 10 minutes. An extraction with phenol/chloroform/isoamyl alcohol is then performed to physically remove the CIAP. Third, a 6-fold excess of CIAP s
