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.