Advertisement

本文章节


 

Alkaline Phosphatase
载体5‘磷酸基团的碱性磷酸酶消解   

引用 收藏 3次提问与回复 分享您的反馈 被引用

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.
登录/注册账号可免费阅读全文
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.
分类
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

Bo-Yeong Jin
Korea Univ
Why do you separate the digestion reaction into two tubes?
2020/7/27 21:41:27 回复
Anaida Walia
Amity Institute of Biotechnology
Hi, Could you please explain why we had to heat inactivate the reaction ?
2016/10/20 9:42:55 回复
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

2016/10/22 2:19:21 回复


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?
2016/8/23 9:24:12 回复
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

2016/8/24 11:23:45 回复