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[Bio101] Alkaline Phosphatase
[Bio101] 载体5‘磷酸基团的碱性磷酸酶消解   

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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.

简介

碱性磷酸酶从载体中除去5'磷酸基团,从而阻止载体的自身连接并促进其它DNA片段连接到载体中。

材料和试剂

  1. 载体DNA
  2. 限制酶(New England Biolabs)
  3. 碱性磷酸酶:小牛肠碱性磷酸酶(CIP)(New England Biolabs,目录号:M0290)或虾碱性磷酸酶(SAP)(Promega公司,目录号:M8201)

程序

  1. 在100μl消化反应中切割载体(5μg)
  2. 将消化反应分成2个管,每个50μl
  3. 加入2.5μlCIP酶或1μlSAP酶至50μl消化(通常NEB消化缓冲液适用于CIP酶,如果没有,则纯化消化并在适当的缓冲液中重悬DNA)。
  4. 在37℃孵育1小时
  5. 热灭活反应在65℃30分钟(CIP)或10分钟(SAP)
  6. 凝胶净化载体 因为CIP去除磷酸基团,所以最好在连接步骤中加入一些ATP。
  • English
  • 中文翻译
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2012). Alkaline Phosphatase. Bio-protocol Bio101: 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
Department of Biology, University of Pennsylvania, USA

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


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
Department of Biology, University of Pennsylvania, USA

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