Team:Tuebingen/Notebook/Protocols/colony-pcr

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<a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: 16px">Back to Protocols</a>
 
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<h3>Reagents</h3>
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<table border="0">
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  <colgroup>
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    <col width="80">
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    <col width="300">
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  <tr>
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    <td style="text-align: center">0.5 µL</td>
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    <td>MgCl2 (c = 25 mM)</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1.0 µL</td>
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    <td>10x Taq Polymerase Buffer</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">0.1 µL</td>
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    <td>dNTPs (2.5 mM each)</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">0.1 µL</td>
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    <td>Oligo forward</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">0.1 µL</td>
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    <td>Oligo reverse</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">7.7 µL</td>
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    <td>Aqua dest.</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">0.1 µL</td>
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    <td>Taq Polymerase (5 U/µL)</td>
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  </tr>
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</table>
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<p>&nbsp;</p>
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<h3>Procedure</h3>
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<ol>
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  <li>Put some PCR-tubes on ice.</li>
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  <li>The given volumes are for one single reaction of 15 µL. When checking more than one colony, repare a master mix for all colonies. Keep that master mix on ice all the time!</li>
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  <li>Transfer 10 µL of master mix in every ice-cold PCR-tube (1 PCR-tube per colony).</li>
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  <li>Usa a pipet tip in order to pick up a colony from a fresh plate and transfer the colony into the reaction mixture inside a PCR-tube.</li>
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  <li>Smear the tip over a new LB plate + antibiotic in order to create new colonies.</li>
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  <li>Run PCR and control success via <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/gelelectrophoresis">gelelctrophoresis</a>.</li>
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  <li>Store plates of successful colony-PCRs at 4 °C.
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</ol>
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<p>&nbsp;</p>
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<a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: 18px">Back to Protocols</a>
</div>
</div>
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Latest revision as of 12:05, 4 October 2013

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Colony-PCR

Reagents

0.5 µL MgCl2 (c = 25 mM)
1.0 µL 10x Taq Polymerase Buffer
0.1 µL dNTPs (2.5 mM each)
0.1 µL Oligo forward
0.1 µL Oligo reverse
7.7 µL Aqua dest.
0.1 µL Taq Polymerase (5 U/µL)

 

Procedure

  1. Put some PCR-tubes on ice.
  2. The given volumes are for one single reaction of 15 µL. When checking more than one colony, repare a master mix for all colonies. Keep that master mix on ice all the time!
  3. Transfer 10 µL of master mix in every ice-cold PCR-tube (1 PCR-tube per colony).
  4. Usa a pipet tip in order to pick up a colony from a fresh plate and transfer the colony into the reaction mixture inside a PCR-tube.
  5. Smear the tip over a new LB plate + antibiotic in order to create new colonies.
  6. Run PCR and control success via gelelctrophoresis.
  7. Store plates of successful colony-PCRs at 4 °C.

 

Back to Protocols


Retrieved from "http://2013.igem.org/Team:Tuebingen/Notebook/Protocols/colony-pcr"