Team:Groningen/protocols/Ligation

From 2013.igem.org

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<div class="mainContent">
<div class="mainContent">
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<h1>Ligation</h1>
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<h2>Ligation Reaction</h2>
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<h3>Materials:</h3>
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<h5>Materials:</h5>
<ul type="square">
<ul type="square">
<li>MQ water</li>
<li>MQ water</li>
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<li>1.5ml tubes</li>
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<li>1.5 ml tubes</li>
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<li>Ligation buffer</li>
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<li>10x T4 Ligation buffer</li>
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<li>Ligase</li>
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<li>T4 Ligase</li>
<li>Insert</li>
<li>Insert</li>
<li>Vector</li>
<li>Vector</li>
</ul>
</ul>
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<h3>Reaction:</h3>
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<h5>Reaction mixture:</h5>
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<table border="1">
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<table id="normal" width="60%">
   <tr>
   <tr>
     <th>Component</th>
     <th>Component</th>
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     <th>20&micro;l</th>
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     <th>20 &micro;l</th>
     <th>Final concentration</th>
     <th>Final concentration</th>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>MilliQ water</td>
     <td>MilliQ water</td>
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     <td>up to 20&micro;l</td>
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     <td>up to 20 &micro;l</td>
     <td></td>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>10 ligation buffer</td>
     <td>10 ligation buffer</td>
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     <td ALIGN=CENTER>2&micro;l</td>
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     <td>2 &micro;l</td>
     <td>1x</td>
     <td>1x</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>Vector</td>
     <td>Vector</td>
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     <td ALIGN=CENTER>x&micro;l</td>
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     <td>x &micro;l</td>
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     <td>20-100ng</td>
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     <td>20-100 ng</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <td>Insert</td>
     <td>Insert</td>
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     <td ALIGN=CENTER>x&micro;l</td>
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     <td>x &micro;l</td>
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     <td>3:1 molar ratio over vector</td>
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     <td>3:1 molar ratio over vector&#42;</td>
   </tr>
   </tr>
   <tr>
   <tr>
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     <td>T4 DNA ligase 5U/&micro;l</td>
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     <td>T4 DNA ligase 5 U/&micro;l</td>
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     <td ALIGN=CENTER>2&micro;l</td>
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     <td>2 &micro;l</td>
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     <td>0.5U/&micro;l</td>
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     <td>0.5 U/&micro;l</td>
   </tr>
   </tr>
</table>  
</table>  
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<br>All the reagents are added following the order listed in the table above. When the reaction mix is prepared, the content is mixed and incubated at room temperature (~22&deg;C) for 20 min.
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<p>&#42; When restricted PCR products are ligated together, a 1:1 molar ratio is used.
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<br>Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
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 +
<br>All the reagents are added following the order listed in the table above [1].  
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<br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22&deg;C) for 20 min.
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 +
 
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<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