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