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Alkaline Phosphatase   

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Alkaline phosphatase removes 5' phosphate groups from vector so that prevents self-ligation of the vector and facilitates the ligation of other DNA fragments into the vector.

Materials and Reagents

  1. Vector DNA
  2. Restriction enzymes (New England Biolabs)
  3. Alkaline phosphatase: Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290 ) or Shrimp Alkaline Phosphatase (SAP) (Promega Corporation, catalog number: M8201 )


  1. Cut the vector (5 μg) in 100 μl digestion reaction.
  2. Separate the digestion reaction to 2 tubes, 50 μl each.
  3. Add 2.5 μl CIP enzyme or 1 μl SAP enzyme to 50 μl digestion (normally NEB digestion buffer is good for CIP enzyme, if not, purify the digestion and resuspend DNA in appropriate buffer).
  4. Incubate at 37 °C for 1 h.
  5. Heat inactivate the reaction at 65 °C for 30 min (for CIP) or 10 min (for SAP).
  6. Gel purify the vector.
    Because CIP removes phosphate group, it is better to add some ATP during ligation step.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2012). Alkaline Phosphatase. Bio-101: e237. DOI: 10.21769/BioProtoc.237.

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Anaida Walia
Amity Institute of Biotechnology
Hi, Could you please explain why we had to heat inactivate the reaction ?
10/20/2016 9:42:55 AM Reply
Fanglian He

Hi Anaida,

It is to inactivate alkaline phosphatase otherwise it will continually remove 5' phosphate groups from other DNA fragments, which would affect subsequent ligation step.


10/22/2016 2:19:21 AM

Juan Carlos Catalan
A big doubt: Im using an Alkaline Phosphatase from Invitrogen, this enzyme has its own buffer dilution, should i using a dilution?
8/23/2016 9:24:12 AM Reply
Fanglian He

Hi Juan,

As said in the protocol, "normally NEB digestion buffer is good for CIP enzyme", so, in my case, I did not use the buffer that comes with CIP enzyme. But, I did not use Alkaline Phosphatase from Invitrogen. So, you may need to check if your digestion buffer is also good for your Alkaline Phosphatase.

Hope this helps.


8/24/2016 11:23:45 AM

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