Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"
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100uL of the bacterial cultures were then plated on LB with or without Amp with different concentrations: 10^-1 10^-2 and 10^-3 | 100uL of the bacterial cultures were then plated on LB with or without Amp with different concentrations: 10^-1 10^-2 and 10^-3 | ||
<br><br> | <br><br> | ||
− | <h1 class="date"> | + | <h1 class="date">August 8th</h1> |
<br><br><b>Results</b> | <br><br><b>Results</b> | ||
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<br>all the product is then purified using the PCR purification protocol described in the wiki. | <br>all the product is then purified using the PCR purification protocol described in the wiki. | ||
<br>the concentration of the DNA is then checked using the nanodrop. | <br>the concentration of the DNA is then checked using the nanodrop. | ||
+ | |||
+ | <h1 class="date">August 13th</h1> | ||
+ | |||
+ | <br><br>We put some <i>E. Coli</i> NEB-Turbo to grow overnight in 2 mL LB, at 37°C with shaking. | ||
+ | |||
+ | <h1 class="date">August 14th</h1> | ||
+ | <br><br>We made our <i>E. Coli</i> NEB-Turbo electrocompetent with the following protocol: | ||
+ | |||
+ | <table style="width:50%"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <ul> | ||
+ | <b>Preparation of ectrocompetent cells protocol</b> | ||
+ | <li>The night before the transformation, start an overnight culture of cells. | ||
+ | <li>5 ml LB Amp.</li></li> | ||
+ | <li>The day of the transformation, dilute the cells 100X. | ||
+ | <li>100 ml LB Amp.</li>< | ||
+ | <li>Grow at 30°C for about 90 minutes.</li></li> | ||
+ | <li>Harvest the cells. | ||
+ | <li>When the cells reach an OD600 of between 0.6 and 0.8.</li> | ||
+ | <li>Split the culture into 2x 50 ml falcon tubes, on ice.</li> | ||
+ | <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li></li> | ||
+ | <li>Wash and combine the cells. | ||
+ | <li>Remove the supernatant.</li> | ||
+ | <li>Resuspend the cells in 2x 25 ml of ice cold water.</li> | ||
+ | <li>Combine the volumes in a single 50 ml falcon tube.</li></li> | ||
+ | <li>Wash the cells 2 more times. | ||
+ | <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> | ||
+ | <li>Resuspend in 50 ml of ice cold water.</li> | ||
+ | <li>Repeat.</li></li> | ||
+ | <li>Wash and concentrate the cells for electroporation. | ||
+ | <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> | ||
+ | <li>Resuspend in 1-2 ml of ice cold water.</li> | ||
+ | <li>We will use 200 ul of washed cells per transformation.</li></li> | ||
+ | </ul></td></tr> | ||
+ | </table> | ||
+ | |||
</html> | </html> | ||
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Revision as of 21:31, 14 August 2015