User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/03/29

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Expressing the Double Mutant

  1. Centrifuge the cultures grown overnight at 4500rpm at 4°C for 15 minutes.
  2. Resuspend each of the pellets in 1mL of LB broth.
  3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks previously made with 1L of sterile LB.
  4. Add 1mL 100mg/mL ampicillin to each flask.
    • 100mg/mL ampicillin prepared fresh today with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
  5. Add 1mL of 40mg/ml hemin in dimethylformamide to each of the flasks.
  6. Incubate these flasks at 37°C at 135rpm for about three hours.
  7. Add 1mL of 0.1M IPTG to each flask. Continue to incubate at 37°C at 135rpm for about five more hours.
  8. Centrifuge these cells at 4500rpm at 4°C for 15 minutes.
  9. Resuspend the pellets in 50mM Tris 50mM NaCl, pH ~9.
  10. Place resuspended cells in -80°C freezer for long-term storage.



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