Overview: Alkaline phosphatases (AP) catalyze the removal of 5' phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates. Also they can remove phosphate groups from proteins. These enzymes are most active at alkaline pH - hence the name. There are several sources of alkaline phosphatase that differ in how easily they can be inactivated, such as bacterial alkaline phosphatase (BAP), calf intestinal alkaline phosphatase (CIP) and shrimp alkaline phosphatase. CIP is the most widely used AP and can be effectively destroyed by heat. There are two main applications for alkaline phosphatase in nucleic acid manipulations: 1)Removing 5' phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme. In subsequent ligation reactions, this treatment prevents self-ligation of the vector and thereby greatly facilitates ligation of other DNA fragments into the vector; 2) Removing 5' phosphates from DNA fragments prior to labeling with radioactive phosphate.
Protocols
- Phosphatase treatment of DNA fragments
(Practical Approach Online, Oxford University Press)
Includes treatment with bacterial alkaline phosphatase and calf intestinal phosphatase
http://www.oup.co.uk/pdf/pas/9v1-7-8.pdf
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Alkaline phosphatase treatment of DNA (CIP)
(Schneitz Lab)
http://www.unizh.ch/botinst/Cyto_Website/schneitzL...
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CIP Dephosphorylation of Linearized DNA
(Vesicle Trafficking)
http://cmgm.stanford.edu/~vesicle/protocols/cip.ht...
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De-phosphorylation of DNA
(Dr. Simon Dawson, Department of Biochemistry, University of Nottingham Medical School)
Calf intestinal phosphatase (CIP) treatment of DNA and subsquently purification.
http://www.nottingham.ac.uk/~mbzspd/methods/De-pho...
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Phosphatasing with Shrimp Alkaline Phosphatase (S.A.P.)
(Hedrick Lab, UCSD Cancer Center)
Use to prevent linearized vector plasmid from recircularizing
http://hedricklab.ucsd.edu/Protocol/SAP.html
Added: Tue Dec 10 2002, Hits: 1554, Reviews: 0 Write review Cached