IGEM:UC Berkeley/2006/Knockouts by Electroporation of pOX38 x pKD46

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  • 50 ml of 2YT + Carb + Kan, add 5 ml started culture and (100uL)200mg/ml Arabinose.
  • Grow to OD 1.2 at 30 degrees.
  • Spin about 40 ml in 50 ml conical for 3 minutes @ 8000 rpm. (Use machine in Keasling lab that is cooled.) Dump supernatant.
  • Resuspend in 30 ml cold 10% glycerol.
  • Spin for 3 minutes @ 7500.
  • Dump supernatant.
  • Resuspend in 30 ml cold 10% glycerol. Spin 3 for 3 minutes @ 7500.
  • Dump supernatant, resuspend in remaining volume. You will not pour out all the liquid, so don't worry about leaving enough liquid to resuspend the pellet.
  • Electroporate 50 microliters cells, 1 microliter product
  • Immediately rescue with LB.
  • Rescue for 1 hour @ 37 degrees.
  • Plate
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