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Team:Evry/Notebook/Protocols/Transformation
From 2014.igem.org
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| - | <h4>Transformation of <i>Pseudovibrio</i></h4> | + | <h4>Transformation of <i>Pseudovibrio</i></h4> |
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| - | <li>Place electroporation tanks in ice for 10min<br> | + | <ul> |
| + | <li>Place electroporation tanks in ice for 10min</li><br> | ||
| + | <br> | ||
| + | <li>Take samples of plasmids (25ng-50ng/µL) /!\ Keep them in ice /!\ </li><br> | ||
| + | <br> | ||
| + | <li>Take sample of competent cells /!\ Keep them in ice /!\ </li><br> | ||
| + | <br> | ||
| + | <li>Place 1µL of plasmid in the sample of competent cells</li> <br> | ||
| + | <br> | ||
| + | <li>Transfer the full volume obtained in the electroporation tank</li> <br> | ||
| + | <br> | ||
| + | <li>Place in the electroporator and pulse at 2000V </li><br> | ||
| + | <b>NB:</b> The optimal pulse length is between 5 and 6ms.</li> <br> | ||
| + | <br> | ||
| + | <li>Add 1mL of MB 1X in the 30 seconds following the transformation</li> <br> | ||
| + | <br> | ||
| + | <li>Incubate between 2h and 3h at 30°C with shaking</li> <br> | ||
| + | <br> | ||
| + | <li>Centrifuge to concentrate all cells in the pellet</li> <br> | ||
| + | <br> | ||
| + | <li>Discard the supernatant</li><br> | ||
| + | <br> | ||
| + | <li>Sowed the pellet on selective plates of MB 1X </li><br> | ||
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Revision as of 20:04, 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
- 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
NB: The optimal pulse length is between 5 and 6ms.
