Chemically competent E. coli cells production

Goal

Protocol

1. 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.



SAFETY AND USEFUL RECOMMANDATION
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.



2. 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.



SAFETY AND USEFUL RECOMMANDATION
There is no significant risk in using the device, but before making any measurments, users must know how they have to manipulate it.



3. 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).



SAFETY AND USEFUL RECOMMANDATION
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).



4. Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.

1. Centrifuge for 10 minutes at 3000 rpm and 4°C.



SAFETY AND USEFUL RECOMMANDATION
The centrifuge must be balanced before being switched on. If the device is malfunctioning, it must not be used.



2. 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.


3. 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.


4. Add 100 µL aliquots to your chilled eppendorfs and store at -80°C.