Home Protocols Protocol for Expression Using T7 Express lysY (C3010)

Protocol for Expression Using T7 Express lysY (C3010)

Protocol

  1. Transform expression plasmid into T7 Express lysY. Plate on antibiotic selection plates and incubate overnight at 37°C.
  2. Resuspend a single colony in 10 ml liquid culture with antibiotic.
  3. Incubate at 37°C until OD600 reaches 0.4 - 0.6.
  4. Induce with 40 µl of a 100 mM stock of IPTG (final concentration of 0.4 mM) and induce for 2 hours at 37°C.
  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, inoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C until OD600 reaches 0.4-0.6. Add IPTG to 0.4 mM. Induce 2 hours at 37°C or 15°C overnight.