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  • Expression Using SHuffle (C3029)

    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.