Team:Groningen/protocols/Ligation

From 2013.igem.org

(Difference between revisions)
 
(20 intermediate revisions not shown)
Line 22: Line 22:
<div class="mainContent">
<div class="mainContent">
-
<h1>Ligation</h1>
+
<h2>Ligation Reaction</h2>
-
<h3>Materials:</h3>
+
<h5>Materials:</h5>
<ul type="square">
<ul type="square">
-
<li>H<sub>2</sub>O</li>
+
<li>MQ water</li>
-
<li>1.5ml tubes</li>
+
<li>1.5 ml tubes</li>
-
<li>Ligation buffer</li>
+
<li>10x T4 Ligation buffer</li>
-
<li>Ligase</li>
+
<li>T4 Ligase</li>
<li>Insert</li>
<li>Insert</li>
<li>Vector</li>
<li>Vector</li>
</ul>
</ul>
-
<h3>Reaction:</h3>
+
<h5>Reaction mixture:</h5>
-
<table border = "1">
+
<table id="normal" width="60%">
-
<tr><td>Component</td>
+
  <tr>
-
<td>20&#956;l reaction</td>
+
    <th>Component</th>
-
<td>Final concentration</td>
+
    <th>20 &micro;l</th>
-
</tr>
+
    <th>Final concentration</th>
-
<tr><td>H<sub>2</sub>O</td>
+
  </tr>
-
<td>up to 20&#956;l</td>
+
-
<td></td>
+
-
</tr>
+
-
<tr><td>10x Ligation buffer</td>
+
-
<td>2&#956;l</td>
+
-
<td>1x</td>
+
-
</tr>
+
-
<tr><td>Vector</td>
+
-
<td>1&#956;l</td>
+
-
<td>20-100&#956;g</td>
+
-
</tr>
+
-
<tr><td>Insert</td>
+
-
<td>1&#956;l</td>
+
-
<td>1:1 molar ratio</td>
+
-
</tr>
+
-
<tr><td>T4 DNA Ligase</td>
+
-
<td>0.2&#956;l</td>
+
-
<td>5U/&#956;l</td>
+
-
</tr>
+
-
</table>
+
-
<br>All the reagents are added following the order listed in the table above.
+
  <tr>
-
<br>After the reaction is ready mix the content of the tube and spin it down.
+
    <td>MilliQ water</td>
-
<br>The tubes are incubated for 1h at 37&deg;C.
+
    <td>up to 20 &micro;l</td>
-
<br>
+
    <td></td>
-
<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
+
  </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>
</div>

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