Team:Aachen/Wetlab/Protocol12

Creation of chemical competent E. coli cells

Materials:


Glassware and Equipment:


  • 200 ul pipetman or repeating pipettor

  • Falcon tubes

  • 500 ul Eppendorf tubes, on ice

  • 200 ml conical flask

  • 5 ml pipette

Preparation:


  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.

  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).

  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 ℃ but if you have just made it fresh then put it in an ice bath).

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

    5. All subsequent steps should be carried out at 4 ℃ and the cells should be kept on ice wherever possible

  5. Centrifuge for 10 minutes at 3000 rpm and 4 ℃.

  6. 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.

  7. PResuspend 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.

  8. Add 100 ul aliquots to your chilled eppendorfs and store at − 80 ℃.

  9. Place the cells at -80 ℃

Notes:


  • If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 ul aliquots.

  • It is a good idea to run a positive control on the cells.