Description

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.

Features

  • Suitable for reactions at high temperatures due to its high heat stability.

Applications

  • Removes 5’ and 3’ phosphoryl groups from nucleic acids
  • Prepares templates for 5’ end-labeling
  • Prevents DNA fragments from self-ligating
  • Dephosphorylates proteins

Source

Escherichia coli K12 SW1033/pk 1-5

Unit definition

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.

Storage conditionn

50 mM Tris-HCl (pH7.5), 50% glycerol
Store at -20ºC

Components

E. coli Alkaline Phosphatase (1-3 U/µl)
10 x Buffer

Principle

Typical reaction set up

Component Amount
DNA 0.5-2.5µg
10 x Buffer 10 µl
Alkaline Phosphatase (0.1-1U/µl) 10 µl
Distilled water X µl
Total reaction volume 50 µl

<Reaction condition>

5’ overhanged end: 37ºC, 60 min.
3’ overhanged or blunt end: 60ºC, 60 min.

Note

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.

References

  • 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)