User:Matt Hartings/Notebook/Photosynthesis/2012/06/20

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WT Hemoglobin Expression

  1. Expression continued from yesterday.
  2. In morning, spin down starter cultures (4500rpm for 20min at 4C)
  3. Resuspend cell pellets in fresh broth and divide evenly among 1L expression flasks
  4. Incubate at 37C and shake at 165rpm
  5. When OD(600) reaches 0.6 induce expression by adding 1mL of 0.4M IPTG and 1mL of 40mg/mL hemin in DMF to cultures
  6. Change shaker temperature to 30C
  7. 4 hours after inducing, harvest cells in centrifuge (4500rpm, 30min, 4C)
  8. Resuspend cells in 50mL of 25mM Tris, 50mM NaCl, 1mM EDTA, 0.5mM DTT
  9. Store in -80C Freezer


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