Protocol for Protein Expression Using BL21 (C2530)


  1. Transform expression plasmid into BL21. Plate on antibiotic selection plates and incubate overnight at 37°C.
  2. Resuspend a single colony in liquid culture with antibiotic to produce a starter culture. Inoculate starter culture at a 1:100 dilution into expression media containing antibiotic.
  3. Incubate at 37°C with shaking until OD600 reaches 0.4–0.8.
  4. For most vector systems, induce with 40 or 400 μM IPTG and express protein for 3 hours at 37°C, 5 hours at 30°C or overnight at 16°C or 23°C.
  5. For large scale, inoculate 1 Liter of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C until OD600 reaches 0.4–0.8. Add 40 or 400 μM IPTG and express protein using optimal time/temperature determined in a small scale trial.