IGEM:UC Berkeley/2006/Knockouts

Procedure for Knockouts

1. 50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose
2. Grow to OD=.8 (the higher the better)
3. Spin approx. 40mL in 50mL conical, 3 minutes @ 8000
4. Resuspend 30mL 10% glycerol
5. Spin 3 minutes @ 7500
6. Repeat glycerol suspension (step 4)
7. Dump supernatant, resuspend in remaining liquid (don't add anything in this step)
8. Electroporate 50ul cells and 1ul PCR product
9. Rescue one hour @ 37C
10. Plate

Excising FRT-flanked cassettes with pCP20

1. Grow up a colony of the strain containing the cassette from a freshly restreaked plate
2. Prepare a small scale electrocompetent cell prep, transform with pCP20
3. Plate on Amp, or Cm+Amp, at 30 degrees
4. Next day, use a tip or toothpick to swab some of the colonies, put in 4 mL of 2YT
5. Grow to saturation at 37 degrees
6. Dilute 10^6, plate on LB-only plates at 43 degrees
7. Grow up and spot individual colonies on Amp and the excision marker