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.

General description

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.

Other Notes

Alkaline phosphatase in 5 mM EDTA (pH 8) will be irreversibly heat inactivated at 75 °C for 10 minutes.

Unit Definition

One unit will hydrolyze 1 µmole of p-nitrophenyl phosphate per min at 37 °C.