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Team:Grenoble/Biology/Protocols/Competence
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<h1>Chemically competent <i>E. coli</i> cells production</h1> | <h1>Chemically competent <i>E. coli</i> cells production</h1> | ||
<h2>Goal</h2> | <h2>Goal</h2> | ||
| - | Product <i>E. coli</i> cells which are competent for | + | Product <i>E. coli</i> cells which are competent for transformation. |
</section> | </section> | ||
<br/> | <br/> | ||
| Line 15: | Line 15: | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
Following are the instructions from the | Following are the instructions from the | ||
| - | <a href="http://openwetware.org/wiki/Preparing_chemically_competent_cells">OpenWetWare website</a>. | + | <a href="http://openwetware.org/wiki/Preparing_chemically_competent_cells" target="blank">OpenWetWare website</a>. |
<br/> | <br/> | ||
<br/> | <br/> | ||
| Line 23: | Line 23: | ||
flask. You should aim to dilute the overnight culture by at least 1/100.</li> | flask. You should aim to dilute the overnight culture by at least 1/100.</li> | ||
<br/> | <br/> | ||
| - | <center><table> | + | <center><table class="tableau"> |
| - | <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | + | <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> |
<tr><td><div class="petit">This step must be done under a flow hood. | <tr><td><div class="petit">This step must be done under a flow hood. | ||
| - | The | + | The flow hood must be sterile, therefore, it is recommended to activate the |
| - | + | venting system before opening the flow closet. | |
| - | + | In case of any doubts, use the UV lamp to sterilize the working space.</div></td></tr> | |
| - | In case of any | + | |
</table> | </table> | ||
</center> | </center> | ||
<br/> | <br/> | ||
<li>Grow the diluted culture to an OD<span class="indice">600</span> of 0.2 - 0.5. | <li>Grow the diluted culture to an OD<span class="indice">600</span> of 0.2 - 0.5. | ||
| - | (You will get a very small pellet if you grow 25 mL to OD<span class="indice">600</span> 0.2).</li> | + | (You will get a very small pellet if you grow 25 mL to OD<span class="indice">600</span> 0.2). The OD is checked using a spectrophotometer.</li> |
<br/> | <br/> | ||
| - | <center><table> | + | <center><table class="tableau"> |
| - | <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | + | <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> |
| - | <tr><td><div class="petit">There is no significant risk | + | <tr><td><div class="petit">There is no significant risk in using the device, |
but before making any measurments, users must know how they have to manipulate it.</div></td></tr> | but before making any measurments, users must know how they have to manipulate it.</div></td></tr> | ||
</table> | </table> | ||
| Line 49: | Line 48: | ||
in an ice bath).</li> | in an ice bath).</li> | ||
<br/> | <br/> | ||
| - | <center><table> | + | <center><table class="tableau"> |
| - | <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | + | <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> |
| - | <tr><td><div class="petit"> | + | <tr><td><div class="petit">To prevent the ice conrainer from falling off the workbench, it is |
| - | recommended to place | + | recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack. |
| - | + | It is important to know where manipulations will be achieved in order to apprehend the possible risks. | |
| - | + | Tubes have to be sterile (keep them closed if they are not under the flow cabinet).</div></td></tr> | |
| - | + | ||
</table> | </table> | ||
</center> | </center> | ||
<br/> | <br/> | ||
| - | <li>Split the culture into two | + | <li>Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.</li> |
</ol> | </ol> | ||
<br/> | <br/> | ||
| - | <b>All subsequent steps should be carried out at 4°C and the cells should be kept on ice | + | <b>All subsequent steps should be carried out at 4°C and the cells should be kept on ice as much as possible.</b> |
| - | + | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
| - | <center><table> | + | <center><table class="tableau"> |
| - | <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | + | <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> |
| - | <tr><td><div class="petit"> | + | <tr><td><div class="petit">It is recommended to change gloves every 30 minutes.</div></td></tr> |
| - | + | ||
| - | + | ||
</table> | </table> | ||
</center> | </center> | ||
| Line 77: | Line 72: | ||
<li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li> | <li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li> | ||
<br/> | <br/> | ||
| - | <center><table> | + | <center><table class="tableau"> |
<tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr> | ||
<tr><td><div class="petit">The centrifuge must be balanced before being switched on. | <tr><td><div class="petit">The centrifuge must be balanced before being switched on. | ||
| - | + | If the device is malfunctioning, it must not be used.</div></td></tr> | |
</table> | </table> | ||
</center> | </center> | ||
<br/> | <br/> | ||
<li>Remove supernatant. The cell pellets should be sufficiently solid that you can | <li>Remove supernatant. The cell pellets should be sufficiently solid that you can | ||
| - | just | + | just discard the supernatant if you are careful. Pipette out any remaining media.</li> |
<br/> | <br/> | ||
<li>Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the | <li>Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the | ||
| - | culture volume that you | + | culture volume that you span down. You may need to vortex gently to fully |
resuspend the culture, keep an eye out for small cell aggregates even after | resuspend the culture, keep an eye out for small cell aggregates even after | ||
the pellet is completely off the wall.</li> | the pellet is completely off the wall.</li> | ||
Latest revision as of 08:23, 25 September 2012
Chemically competent E. coli cells production
Goal
Product E. coli cells which are competent for transformation.Protocol
Following are the instructions from the OpenWetWare website.- Grow a 5 mL overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50 mL of fresh LB media in a 200 mL conical flask. You should aim to dilute the overnight culture by at least 1/100.
- Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25 mL to OD600 0.2). The OD is checked using a spectrophotometer.
- Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
- Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.
This step must be done under a flow hood.
The flow hood must be sterile, therefore, it is recommended to activate the
venting system before opening the flow closet.
In case of any doubts, use the UV lamp to sterilize the working space. |
There is no significant risk in using the device,
but before making any measurments, users must know how they have to manipulate it. |
To prevent the ice conrainer from falling off the workbench, it is
recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack.
It is important to know where manipulations will be achieved in order to apprehend the possible risks.
Tubes have to be sterile (keep them closed if they are not under the flow cabinet). |
All subsequent steps should be carried out at 4°C and the cells should be kept on ice as much as possible.
It is recommended to change gloves every 30 minutes. |
- Centrifuge for 10 minutes at 3000 rpm and 4°C.
- Remove supernatant. The cell pellets should be sufficiently solid that you can just discard the supernatant if you are careful. Pipette out any remaining media.
- Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you span down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
- Add 100 µL aliquots to your chilled eppendorfs and store at -80°C.
SAFETY AND USEFUL RECOMMANDATION |
|---|
The centrifuge must be balanced before being switched on.
If the device is malfunctioning, it must not be used. |
