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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 (50µg/ml) (final concentrations). The
digestion is allowed to proceed at 56°C for 30 minutes at which time the proteinase K can
be inactivated by heating at 70°C for 10-15 minutes. Second, EDTA can be added to a final
concentration of 5mM and the reaction heated at 75°C 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 stop buffer (consisting of 10mM Tris-HCl (pH 7.5), 1mM EDTA, 200mM
NaCl and 0.5% SDS) is added to the dephosphorylation reaction, which is then extracted
with phenol/chloroform/isoamyl alcohol followed by ethanol precipitation of the
dephosphorylated DNA. |
