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 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



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).



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.



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
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.



4. Split the culture into two 50ml 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. In case of dysfunction of the device, it must not be used.



2. 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.


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.