Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"
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<b>Preparation of ectrocompetent cells protocol</b> | <b>Preparation of ectrocompetent cells protocol</b> | ||
<li>The night before the transformation, start an overnight culture of cells. | <li>The night before the transformation, start an overnight culture of cells. | ||
− | <li>5 ml LB Amp.</li></li> | + | <li>5 ml LB Amp.</li></li></ul> |
<li>The day of the transformation, dilute the cells 100X. | <li>The day of the transformation, dilute the cells 100X. | ||
− | <li>100 ml LB Amp.</li> | + | <ul><li>100 ml LB Amp.</li> |
− | <li>Grow at 30°C for about 90 minutes.</li></li> | + | <li>Grow at 30°C for about 90 minutes.</li></li></ul> |
<li>Harvest the cells. | <li>Harvest the cells. | ||
− | <li>When the cells reach an OD600 of between 0.6 and 0.8.</li> | + | <ul><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>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>Centrifuge at 4 °C for 10 min at 4000 rpm.</li></li></ul> |
<li>Wash and combine the cells. | <li>Wash and combine the cells. | ||
− | <li>Remove the supernatant.</li> | + | <ul><li>Remove the supernatant.</li> |
<li>Resuspend the cells in 2x 25 ml of ice cold water.</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>Combine the volumes in a single 50 ml falcon tube.</li></li></ul> |
<li>Wash the cells 2 more times. | <li>Wash the cells 2 more times. | ||
− | <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> | + | <ul><li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> |
<li>Resuspend in 50 ml of ice cold water.</li> | <li>Resuspend in 50 ml of ice cold water.</li> | ||
− | <li>Repeat.</li></li> | + | <li>Repeat.</li></li></ul> |
<li>Wash and concentrate the cells for electroporation. | <li>Wash and concentrate the cells for electroporation. | ||
− | <li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> | + | <ul><li>Centrifuge at 4 °C for 10 min at 4000 rpm.</li> |
<li>Resuspend in 1-2 ml of ice cold water.</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> | + | <li>We will use 200 ul of washed cells per transformation.</li></li></ul> |
</ul></td></tr> | </ul></td></tr> | ||
</table> | </table> |
Revision as of 21:32, 14 August 2015