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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<li>Place electroporation tanks in ice for 10min
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<li>Take samples of plasmids (25ng-50ng/µL)    /!\ Keep them in ice /!\ <br>
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<li>Place electroporation tanks in ice for 10min</li>
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<li>Take sample of competent cells  /!\ Keep them in ice /!\ <br>
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<li>Take samples of plasmids (25ng-50ng/µL)    /!\ Keep them in ice /!\ </li>
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<li>Place 1µL of plasmid in the sample of competent cells <br>
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<li>Take sample of competent cells  /!\ Keep them in ice /!\ </li>
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<li>Transfer the full volume obtained in the electroporation tank <br>
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<li>Place 1µL of plasmid in the sample of competent cells</li>  
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<li>Place in the electroporator and pulse at 2000V <br>
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<li>Transfer the full volume obtained in the electroporation tank</li>  
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<b>NB:<b>  The optimal pulse length is between 5 and 6ms. <br>
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<li>Place in the electroporator and pulse at 2000V </li>
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<li>Add 1mL of MB 1X in the 30 seconds following the transformation <br>
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<b>NB:</b>  The optimal pulse length is between 5 and 6ms.</li>
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<li>Incubate between 2h and 3h at 30°C with shaking <br>
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<li>Add 1mL of MB 1X in the 30 seconds following the transformation</li>
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<li>Centrifuge to concentrate all cells in the pellet <br>
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<li>Incubate between 2h and 3h at 30°C with shaking</li>
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<li>Discard the supernatant<br>
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<li>Centrifuge to concentrate all cells in the pellet</li>
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<li>Sowed the pellet on selective plates of MB 1X <br>
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<li>Discard the supernatant</li>
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<li>Sowed the pellet on selective plates of MB 1X </li>
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Latest revision as of 00:48, 18 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

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