IGEM:UC Berkeley/2006/Knockouts

From OpenWetWare
Revision as of 10:57, 20 July 2006 by Mfleming (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

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