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Team:Grenoble/Biology/Protocols/Competence
From 2012.igem.org
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<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 | ||
wherever possible.</b> | wherever possible.</b> | ||
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<br/> | <br/> | ||
<center><table> | <center><table> | ||
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<ol> | <ol> | ||
<li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li> | <li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li> | ||
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| + | <center><table> | ||
| + | <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. | ||
| + | In case of dysfunction of the device, it must not be used.</div></td></tr> | ||
| + | </table> | ||
| + | </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 | ||
Revision as of 06:30, 27 August 2012
Chemically competent E. coli cells production
Goal
Product E. coli cells which are competent for a chemical 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).
- 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 50ml falcon tubes and incubate on ice for 10 min.
SAFETY AND USEFUL RECOMMANDATION |
|---|
This step must be done into a flow hood.
The flood hood must be sterile.
Therefore it is recommended to activate the
ventilator before taking the curtain down.
In case of any doubt, it can be interesting to use the UV lamp |
SAFETY AND USEFUL RECOMMANDATION |
|---|
There is no significant risk by using the device,
but before wanting to use it, users must know how they have to manipulate it. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
In order to reduce the risk of ice that could fall, it is
recommended to place the back near the place manipulations and in case of any move,
it is better to take eppendorfs in an eppendorf rack. That is why it is important to
know where manipulations will be done. Tubes must also be sterile and nowhere be open
except into the flow hood. |
All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible.
SAFETY AND USEFUL RECOMMANDATION |
|---|
All manipulations are down in a cold atmosphere. That is why ice is used.
The ice has to be correctly put in backs that should not be moved, to prevent its fall. It is also
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 pour off 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 spun 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.
In case of dysfunction of the device, it must not be used. |
