Team:USTC CHINA/Notebook/Protocols/Ligation

From 2013.igem.org

(Difference between revisions)
(Created page with "{{USTC-China/hidden}} <html> <head> <link rel="stylesheet" type="text/css" href="https://2013.igem.org/Team:USTC_CHINA/main.css?action=raw&ctype=text/css" /> </head> <body backgro...")
Line 53: Line 53:
<div class="content" align="center">
<div class="content" align="center">
<div class="conbar1">
<div class="conbar1">
-
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
+
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a>&gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Ligation">protocols Ligation</a></div></div>
<div class="conbar2">
<div class="conbar2">
<div class="leftbar" align="left">
<div class="leftbar" align="left">
<div class="bassic-bar">
<div class="bassic-bar">
-
<h1>Gel Extraction</h1>
+
<h1>Ligation</h1>
-
<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
+
<p><span>Performed with TaKaRa Ligation Kit (Solution I)</span></br>
-
Protocol
+
1. System</br>
-
1. Excise gel slice containing DNA fragment of interest.
+
</br>
-
2. Add 3×sample volume of Buffer DE-A.
+
<table width="580" border="2">
-
Incubate at 75° C for 15-20 min or until gel melts completely.
+
  <tr>
-
Add 0.5 × Buffer DE-A volume of Buffer DE-B.
+
    <td></td>
-
<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
+
    <td>Volume (μl)</td>
-
3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
+
  </tr>
-
+
  <tr>
-
4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
+
    <td>Solution I</td>
-
Repeat wash with Buffer W2
+
    <td>5</td>
-
Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
+
  </tr>
-
+
  <tr>
-
5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
+
    <td>DNA (fragment + carrier)</td>
 +
    <td>5</td>
 +
  </tr>
 +
</table>
 +
</br>
 +
fragment : carrier (radio in mol)= 3:1~10:1</br>
 +
carrier≥0.03 pmol</br>
 +
2. Gently vortex the samples and spin down.</br>
 +
3. Incubate at 16°C in a low temperature water thermostat for at least 3h.</br>
 +
 
   
   

Revision as of 17:10, 25 September 2013

Ligation

Performed with TaKaRa Ligation Kit (Solution I)
1. System

Volume (μl)
Solution I 5
DNA (fragment + carrier) 5

fragment : carrier (radio in mol)= 3:1~10:1
carrier≥0.03 pmol
2. Gently vortex the samples and spin down.
3. Incubate at 16°C in a low temperature water thermostat for at least 3h.

notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Ligation"