Herskowitz Lab Protocol
From : Megan Grether/Corrie Detwiler

FACS Protocol

  1. grow > 0.5ml cells to OD 0.5-2
  2. add 1ml 100% EtOH to 0.5ml cells
  3. rotate a minimum of 1hr (max 1 week)
  4. pellet 1 minute
  5. wash with 1ml Tris 50mM pH7.5
  6. pellet 1 minute
  7. add RNase 1mg/ml 0.5ml -> vortex
  8. incubate 37 deg for 1-2 hours, or 4 deg overnight (up to 40hr)
  9. add 1ul of 20mg/ml Proteinase K in water (0.04mg/ml final)
  10. vortex
  11. incubate 1 hour 50-55 deg. (temperature, time is critical)
  12. pellet
  13. wash with 1X PBS (1ml)
  14. sonicate again
  15. pellet and resuspend in the dark with 200ul of 100ug/ml propidium iodide.[in PBS and azide].
  16. incubate 1 hour - days
  17. dilute 100ul to 0.5ml in PBS in a falcon 2052 tube.

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