• Plate of cells to be made competent
• TSS buffer
• LB media
• Ice

• Falcon tubes
• 500μl Eppendorf tubes, on ice
• 200ml conical flask
• 200μl pipetman or repeating pipettor
• 5ml pipette

1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
2. Grow the diluted culture to an OD₆₀₀ of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD₆₀₀ 0.2)
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^oC but if you have just made it fresh then put it in an ice bath).
4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4^oC and the cells should be kept on ice wherever possible

1. Centrifuge for 10 minutes at 3000 rpm and 4^oC.
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^oC.
   • The original paper [1] suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at − 80^oC also seems to work well (Jkm)
   • If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence.
5. It is a good idea to run a positive control on the cells.
   • The Endy Lab is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out here.