IGEM:UC Berkeley/2006/Electrocompetent cells

50 mL scale Electrocompetent cells

Combine 50 mL of LB + antibiotic and 2 mL of saturated overnight cell culture
Place in 37 C shaker until OD reaches between 0.8 and 1.2

1. 45 mL of solution (described above)
2. Centrifuge at 8000 RPM for 3 minutes
3. Save pellet, dispose of excess fluid
4. Fill to 30 mL with 10% glycerol
5. Vortex
6. Centrifuge at 7500 RPM for 3 minutes
7. Save pellet, dispose of excess fluid
8. Fill to 30 mL with 10% glycerol
9. Vortex
10. Centrifuge at 7500 RPM for 3 minutes
11. Save pellet, dispose of excess fluid

1 mL scale Electrocompetent cells

Grow 5 mL of bacteria in LB or 2YT + antibiotics to OD=~1.0
Do all subsequent steps on ice.
10% glycerol needs to be very clean, sterile, and cold.

1. Cool on ice
2. transfer 1 mL to an eppendorf, spin 20 sec at full speed
3. dump supe, resuspend in 1 mL 10% glycerol
4. spin, dump supe, resuspend in 1 mL 10% glycerol
5. spin, dump supe, resuspend in 100 uL of 10% glycerol

Ready for electroporation.