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  • Expression Using NEB Express (C2523)

    Protocol

    1. Transform expression plasmid into NEB Express. 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.