SampleInduction Protocol (for reference only) Inoculatea single colony from a freshly streaked plate into 5 ml of LB medium containingthe appropriate antibiotic for theplasmid andhost strain. 2. Incubate with shakingat 200 rpm at 37℃ overnight. 3. Inoculate50 ml of LB medium containing the appropriateantibiotic with 0.5ml of the overnight culture prepared in step 2(usethe 500 ml triangular flask as thecontainer would bebetter). 4. Incubate with shakingat 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8. 5. (Optional)Pipet1ml of the cultures into clean microcentrifuge tubes and place the tubes on ice until needed for gel analysis or storage at -20℃. These will serve as thenon-induced control samples. 6. AddIPTG to a final concentration of 1 mM. Optimal time for induction of thetarget protein may vary from 2-16 hours, depending on the protein. 7. Incubatewithshakingat120rpmat37℃ for3-4hours.Todeterminetheoptimaltimeforinduction of the target protein, it is recommended that a time course experiment beperformed varying the induction from 2-16 hours. 8. Place theculture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10 minutes at 4℃. 9. Remove the supernatant andstore the cell pellet at -20℃ (storage at lower temperatures is also acceptable).
IPTG Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside; Isopropyl-β-D-thiogalactopyranoside) by dissolving 2.38 g of IPTG in dd water andadjust the final volume to 10 ml. Filter sterilize before use.