Team:Macquarie Australia/IPTG

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Protein Expression
IPTG Induction


  1. Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol.
  2. Screen plates for transformant colonies and place in 5mL of LB broth with appropriate antibiotic concentration at 37oC with shaking until OD600¬ is 2.0 – 3.0.
  3. Take whole volume and place in 45mL of LB broth in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37oC with shaking until OD600 is 0.5-0.7.
  4. Transfer 1M IPTG in a 1:1000 dilution into flask. Incubate at 15oC with shaking overnight.
  5. Cell pellets were collected through centrifugation at 12,000 rpm for 5 mins.
  6. Cell pellets were resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.


Auto Induction


  1. Start with glycerol stock or freshly transformed plate.
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  2. Inoculate 2ml starter culture with a single colony from plate, grow at 37oC with vigorous shaking to OD600 ~ 1.
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  3. Transfer to a larger culture (50ml of ZYM-5052 medium in 250ml flask) and grow at room temperature (or 18o C) overnight ~ 200rpm. For small scale, same starter flask can be grown at room temperature for ~24h. Amount of protein expressed peaks at OD600 ~ 8-10.
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  4. Spin down 5000 x g for 20 min at 4oC, discard supernatant.
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  5. Resuspend pellet in 1/10th of the original volume.
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  6. To run on NuPage Bis-Tris gels, make sample mixture which contains: 20 µL resuspended lysate, 20 µL milli-Q water, 16µL NuPage LDS sample buffer, and 5µL 1M DTT.
  7. Boil for 20 min, then spin down max speed for 20 min. Load 10-20µL of supernatant on gels.
  8. Run gel at 150V for 1.5h, then fix with Coomassie Fixing solution for 10 min, stain with Coomassie Stain Solution for 5 min and destain overnight with Destain Solution.