Home Protocols Expression Using SHuffle (C3030)

Expression Using SHuffle (C3030)

Protocol

  1. Transform expression plasmid into SHuffle. Plate on antibiotic selection plates and incubate 24 hours at 30°C.
  2. Resuspend a single colony in 10 ml liquid medium with antibiotic.
  3. Incubate at 30°C until OD600 reaches 0.4–0.8.
  4. Add the appropriate inducer, e.g. 40 μl of a 100 mM stock of IPTG. Incubate for 4 hours at 30°C or 16°C overnight.
  5. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction alone.
  6. For large scale, innoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 30°C until reaches 0.4–0.8. Add the appropriate inducer, e.g. IPTG to 0.4 mM. Induce 4 hours or 16°C overnight.