Team:Groningen/protocols/Ligation

From 2013.igem.org

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<h2>Ligation Reaction</h2>
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<h5>Materials:</h5>
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<ul type="square">
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<li>MQ water</li>
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<li>1.5 ml tubes</li>
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<li>10x T4 Ligation buffer</li>
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<li>T4 Ligase</li>
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<li>Insert</li>
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<li>Vector</li>
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</ul>
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<h5>Reaction mixture:</h5>
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<table id="normal" width="60%">
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  <tr>
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    <th>Component</th>
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    <th>20 &micro;l</th>
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    <th>Final concentration</th>
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  </tr>
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  <tr>
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    <td>MilliQ water</td>
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    <td>up to 20 &micro;l</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>10 ligation buffer</td>
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    <td>2 &micro;l</td>
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    <td>1x</td>
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  </tr>
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  <tr>
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    <td>Vector</td>
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    <td>x &micro;l</td>
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    <td>20-100 ng</td>
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  </tr>
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  <tr>
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    <td>Insert</td>
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    <td>x &micro;l</td>
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    <td>3:1 molar ratio over vector&#42;</td>
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  </tr>
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  <tr>
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    <td>T4 DNA ligase 5 U/&micro;l</td>
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    <td>2 &micro;l</td>
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    <td>0.5 U/&micro;l</td>
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  </tr>
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</table>
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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>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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<p>[1] http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf
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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