Team:Tuebingen/Notebook/Protocols/ligation

From 2014.igem.org

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<div id="TueContent">
<div id="TueContent">
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<h1>Protocols</h1>
<h3>DNA-Ligation</h3>
<h3>DNA-Ligation</h3>
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<h4>Reagents</h4>
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<table border="0">
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  <colgroup>
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    <col width="100">
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    <col width="300">
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  </colgroup>
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  <tr>
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    <td style="text-align: center">20-50 ng</td>
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    <td><a href="https://2014.igem.org/Team:Tuebingen/Notebook/Protocols/restriction">linearized vector</a></td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1 µL</td>
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    <td>10x ligation buffer</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1 µL</td>
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    <td>T4 ligase</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1 µL</td>
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    <td>T4 ligase</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1 µL</td>
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    <td>ATP</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">100-200 ng</td>
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    <td>Insert</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">to 10 µL</td>
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    <td>H<sub>2</sub>O</td>
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  </tr>
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</table>
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<p>&nbsp;</p>
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<h4>Procedure</h4>
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<ol>
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  <li> Mix all reagents in a PCR-tube and if necessary add to 10 μl with H<sub>2</sub>O.</li>
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  <li> Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.</li>
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  <li> Heat inactivate the enzymes for 5 min at 70 °C.</li>
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  <li> Store at -20 °C.</li>
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</ol>
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</div>
</div>

Latest revision as of 15:31, 17 October 2014


Protocols

DNA-Ligation

Reagents

20-50 ng linearized vector
1 µL 10x ligation buffer
1 µL T4 ligase
1 µL T4 ligase
1 µL ATP
100-200 ng Insert
to 10 µL H2O

 

Procedure

  1. Mix all reagents in a PCR-tube and if necessary add to 10 μl with H2O.
  2. Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.
  3. Heat inactivate the enzymes for 5 min at 70 °C.
  4. Store at -20 °C.

Retrieved from "http://2014.igem.org/Team:Tuebingen/Notebook/Protocols/ligation"