Richard Lab:Preparing electrocompetent cells
This protocol is for the preparation of electro-competent E. coli cells which are used for electroporation in the Richard Lab.
The consensus protocol should be consulted if deviating from the procedure outlined here.
Procedure
1. Inoculate 5mL LB Lennox medium and grow overnight at 37°C with rotation.
2. Add the 5mL overnight culture to 200mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. (3 hours)
3. Autoclave the following
- 200 mL LB Lennox or SOB
- 400 mL Millipore Water
- 50 mL 10% glycerol
- 200 mL LB Lennox or SOB
4. Prechill the following:
- Autoclaved water
- Autoclaved Glycerol solution
- Four 50ml centrifuge tubes
- Four 15ml centrifuge tubes
- Twenty microcentrifuge tubes
- Autoclaved water
5. Fast cool the centrifuge with the correct rotor to 4°C.
6. Pour the log phase culture into four 50 mL centrifuge tubes.
7. Place the tubes on ice for 30 minutes.
8. Centrifuge for 10 mins at 2000g at 4°C.
9. Remove supernatant and gently resuspend pellets with 40ml ice-cold sterile water.
11. Centrifuge for 15 mins at 2000g at 4°C.
12. Remove supernatant and gently resuspend pellets in 20ml cold sterile water.
13. Hold on ice for 30 minutes.
14. Centrifuge for 15 mins at 2000g at 4°C.
15. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.
16. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.
17. Centrifuge for 15 mins at 1500g at 4°C.
18. Remove the supernatant and add 500 μl of 10% glycerol.
19. Aliquot 100 μL per tube (tubes on ice).
20. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.
Notes
- The Ice-cold thing is really important
- It is usually helpfull to have two people for the last steps of this protocol, that way one person can pipet 100μL of cell suspension into a tube, and the other person can dip the tube in liquid nitrogen. This saves you from having to keep all those centrifuge tubes on ice (Which is a real pain in the ass)
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