Team:Evry/Notebook/Protocols/Transformation

From 2014.igem.org

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<h4>M9 - CASA +3%NaCl</h4>
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<h4>Transformation of <i>Pseudovibrio<i></h4>
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<br>
<br>
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<li>Place electroporation tanks in ice for 10min
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<li>Place electroporation tanks in ice for 10min<br>
 +
<br>
<li>Take samples of plasmids (25ng-50ng/µL)    /!\ Keep them in ice /!\ <br>
<li>Take samples of plasmids (25ng-50ng/µL)    /!\ Keep them in ice /!\ <br>
 +
<br>
<li>Take sample of competent cells  /!\ Keep them in ice /!\ <br>
<li>Take sample of competent cells  /!\ Keep them in ice /!\ <br>
 +
<br>
<li>Place 1µL of plasmid in the sample of competent cells <br>
<li>Place 1µL of plasmid in the sample of competent cells <br>
 +
<br>
<li>Transfer the full volume obtained in the electroporation tank <br>
<li>Transfer the full volume obtained in the electroporation tank <br>
 +
<br>
<li>Place in the electroporator and pulse at 2000V <br>
<li>Place in the electroporator and pulse at 2000V <br>
<b>NB:</b>  The optimal pulse length is between 5 and 6ms. <br>
<b>NB:</b>  The optimal pulse length is between 5 and 6ms. <br>
 +
<br>
<li>Add 1mL of MB 1X in the 30 seconds following the transformation <br>
<li>Add 1mL of MB 1X in the 30 seconds following the transformation <br>
 +
<br>
<li>Incubate between 2h and 3h at 30°C with shaking <br>
<li>Incubate between 2h and 3h at 30°C with shaking <br>
 +
<br>
<li>Centrifuge to concentrate all cells in the pellet <br>
<li>Centrifuge to concentrate all cells in the pellet <br>
 +
<br>
<li>Discard the supernatant<br>
<li>Discard the supernatant<br>
 +
<br>
<li>Sowed the pellet on selective plates of MB 1X <br>
<li>Sowed the pellet on selective plates of MB 1X <br>
<br>
<br>

Revision as of 11:57, 17 October 2014

Transformation of Pseudovibrio

  • Place electroporation tanks in ice for 10min

  • Take samples of plasmids (25ng-50ng/µL) /!\ Keep them in ice /!\

  • Take sample of competent cells /!\ Keep them in ice /!\

  • Place 1µL of plasmid in the sample of competent cells

  • Transfer the full volume obtained in the electroporation tank

  • Place in the electroporator and pulse at 2000V
    NB: The optimal pulse length is between 5 and 6ms.

  • Add 1mL of MB 1X in the 30 seconds following the transformation

  • Incubate between 2h and 3h at 30°C with shaking

  • Centrifuge to concentrate all cells in the pellet

  • Discard the supernatant

  • Sowed the pellet on selective plates of MB 1X

    • Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Protocols/Transformation"