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