E. coli alkaline phosphatase catalyzes the removal of 5’-phosphate groups from DNA, RNA, and ribo- and deoxyribonucleoside triphosphates. It can be used to prevent self-ligation of vectors because alkaline phosphate-treated DNA fragments lack the 5’-phosphoryl termini required for the actions of DNA ligases.
- Suitable for reactions at high temperatures due to its high heat stability.
- Removes 5’ and 3’ phosphoryl groups from nucleic acids
- Prepares templates for 5’ end-labeling
- Prevents DNA fragments from self-ligating
- Dephosphorylates proteins
Escherichia coli K12 SW1033/pk 1-5
One unit is defined as the amount of enzyme that will dephosphorylate 1µmole of p-Nitrophenyl phosphate with 1 M Tris-HCl (pH 8.0) in 1 min at 25ºC.
50 mM Tris-HCl (pH7.5), 50% glycerol
Store at -20ºC
E. coli Alkaline Phosphatase (1-3 U/µl)
10 x Buffer
Typical reaction set up
|10 x Buffer||10 µl|
|Alkaline Phosphatase (0.1-1U/µl)||10 µl|
|Distilled water||X µl|
|Total reaction volume||50 µl|
5’ overhanged end: 37ºC, 60 min.
3’ overhanged or blunt end: 60ºC, 60 min.
This enzyme can not be inactivated by heating. The reacted DNA fragments should be purified by phenol-chloroform extraction or DNA fragment purification kits (e.g., MagExtractor-PCR and Clean up, code no. NPK-601) prior to ligation.
- T. W. Reid and I. B. Willson, The Enzymes（3rd Ed.), 4: 373-416 (1971)
- A. Garen and C. Levinthal, A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli.
I. Purification and characterization of alkaline phosphatase. Biochem. Biophys. Acta., 38: 470-483 (1960)
- K. Yoda, Y. Kikuchi, M. Yamasaki and G. Tamura, Cloning of the structural gene of alkaline phosphatase of Escherichia coli K12. Agric.
Biol. Chem., 44: 1213-1214 (1980)