Team:Groningen/protocols/Ligation

From 2013.igem.org

(Difference between revisions)
(Created page with "dfbdf")
 
(23 intermediate revisions not shown)
Line 1: Line 1:
-
dfbdf
+
<html>
 +
 
 +
<head>
 +
<meta http-equiv="X-UA-Compatible" content="IE=EDGE" charset="utf-8" />
 +
<link rel="stylesheet" href="https://2013.igem.org/Team:Groningen/CSS/DefaultPage?action=raw&ctype=text/css" type="text/css" />
 +
</head>
 +
<body>
 +
 
 +
</html> {{:Team:Groningen/Templates/DefaultHeader}}<html>
 +
 
 +
 
 +
<div class="colmask threecol">
 +
 
 +
<span class="navigation">
 +
    </html>{{:Team:Groningen/Templates/Navigationbar}}<html>
 +
</span>
 +
 
 +
<span class="widgets">
 +
    </html>{{:Team:Groningen/Templates/facebook}}<html>
 +
</span>
 +
 
 +
<div class="mainContent">
 +
 
 +
<h2>Ligation Reaction</h2>
 +
 
 +
<h5>Materials:</h5>
 +
<ul type="square">
 +
<li>MQ water</li>
 +
<li>1.5 ml tubes</li>
 +
<li>10x T4 Ligation buffer</li>
 +
<li>T4 Ligase</li>
 +
<li>Insert</li>
 +
<li>Vector</li>
 +
</ul>
 +
 
 +
<h5>Reaction mixture:</h5>
 +
<table id="normal" width="60%">
 +
  <tr>
 +
    <th>Component</th>
 +
    <th>20 &micro;l</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MilliQ water</td>
 +
    <td>up to 20 &micro;l</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>10 ligation buffer</td>
 +
    <td>2 &micro;l</td>
 +
    <td>1x</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Vector</td>
 +
    <td>x &micro;l</td>
 +
    <td>20-100 ng</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Insert</td>
 +
    <td>x &micro;l</td>
 +
    <td>3:1 molar ratio over vector&#42;</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>T4 DNA ligase 5 U/&micro;l</td>
 +
    <td>2 &micro;l</td>
 +
    <td>0.5 U/&micro;l</td>
 +
  </tr>
 +
 
 +
</table>
 +
 
 +
<p>&#42; When restricted PCR products are ligated together, a 1:1 molar ratio is used.
 +
 
 +
<br>All the reagents are added following the order listed in the table above [1].
 +
<br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22&deg;C) for 20 min.
 +
 
 +
 
 +
<p>[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
 +
 
 +
</div>
 +
 
 +
</html> {{:Team:Groningen/Templates/DefaultFooter}}<html>
 +
 
 +
</body>
 +
</html>

Latest revision as of 23:36, 4 October 2013

Ligation Reaction

Materials:
  • MQ water
  • 1.5 ml tubes
  • 10x T4 Ligation buffer
  • T4 Ligase
  • Insert
  • Vector
Reaction mixture:
Component 20 µl Final concentration
MilliQ water up to 20 µl
10 ligation buffer 2 µl 1x
Vector x µl 20-100 ng
Insert x µl 3:1 molar ratio over vector*
T4 DNA ligase 5 U/µl 2 µl 0.5 U/µl

* When restricted PCR products are ligated together, a 1:1 molar ratio is used.
All the reagents are added following the order listed in the table above [1].
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.

[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf