Navigate this Article


 

Alkaline Phosphatase   

How to cite Favorites 3 Q&A Share your feedback Cited by

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.
Please login or register for free to view full text
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.
Categories
Q&A
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at eb@bio-protocol.org.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Bo-Yeong Jin
Korea Univ
Why do you separate the digestion reaction into two tubes?
7/27/2020 9:41:27 PM Reply
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
Bio-protocol

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

10/22/2016 2:19:21 AM Reply


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?
8/23/2016 9:24:12 AM Reply
Fanglian He
Bio-protocol

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

8/24/2016 11:23:45 AM Reply


We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.