Alkaline phosphatase is a phosphomonoesterase that catalyzes the cleavage of terminal phosphates from nucleic acids. The free 5′-OH can be phosphorylated with polynucleotide kinase and γ32P-ATP to produce γ32P-end-labeled nucleic acids. Linearized cloning vectors can be prevented from recircularizing by dephosphorylation with alkaline phosphatase.
Application
Commonly used to remove the 5′-terminal phosphate from nucleic acids during molecular cloning reactions.
Components
Alkaline phosphatase is provided in a solution of 10 mM Tris-HCl (pH 8.2), 50 mM KCl, 1 mM MgCl2, and 0.1 mM ZnCl2, in 50% (w/v) glycerol. 10X CIP Buffer, Product Number C3225, is included.
Unit Definition
One unit will hydrolyze 1 μmole of p-nitrophenyl phosphate per min at 37 °C.
Other Notes
Alkaline phosphatase in 5 mM EDTA (pH 8) will be irreversibly heat inactivated at 75 °C for 10 minutes.
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CIP is used to remove 5’-phosphate groups from DNA, RNA and both ribo and deoxy-ribonucleoside triphosphates. Detailed protocol on how to dephosphorylate DNA.
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