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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.

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[Bio101] Alkaline Phosphatase

Molecular Biology > DNA > DNA cloning

[Abstract] 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)

Procedure

  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.


How to cite: He, F. (2012). Alkaline Phosphatase. Bio-protocol Bio101: e237. DOI: 10.21769/BioProtoc.237; Full Text



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10/20/2016 9:42:55 AM  

Anaida Walia
Amity Institute of Biotechnology

Hi, Could you please explain why we had to heat inactivate the reaction ?

10/22/2016 2:19:21 AM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

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.

--Fanglian

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8/23/2016 9:24:12 AM  

Juan Carlos Catalan
INSP

A big doubt: Im using an Alkaline Phosphatase from Invitrogen, this enzyme has its own buffer dilution, should i using a dilution?

8/24/2016 11:23:45 AM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

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.

--Fanglian

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