Lab Protocols/Protein expression in E. coli BL21 DE3 cells

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Protein expression in BL21 (DE3) Tuner cells

  1. Grow overnight strains, with antibiotic where required in LB overnight at 37C. Include controls of BL21 cells that contain plasmid without the gene of interest, and BL21 cells alone without plasmid
  2. Next day, measure the OD600 of the cultures (should be around OD of 3-4) and calculate required volume to add to 10ml of LB culture in new tubes (volume in ul = 0.05*10,000/measured OD).
  3. There will need to be N tubes in the experiment, with each sample to be tested being grown with or without IPTG induction. To each tube, add the required volume of overnight culture to 10ml LB and add required antibiotic where needed (50ul in 10ml is typical on Level 2 CBCB)
  4. Incubate for 1.5-2hrs at 37C shaking on the platform
  5. Measure the OD600 after this time – we need it to be around 0.4 to 0.6
  6. To the appropriate tubes, defrost and add 1M IPTG to final concentration of 1mM (10ul in 10ml; stocks are in the Level 2 -20C freezer)
  7. Incubate for a further three hours at 37C
  8. Remove 2ml of culture and transfer to 2ml spin tubes
  9. Centrifuge the culture for 2mins at 4C. Remove supernatant transfer to eppendorf tube. Do not disturb the pellet.
  10. Store the pellets and supernatents in a freezer box at -20C