Yu:Harvest cells

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Harvest Yeast Cells

  1. Grow cell culture to late-log overnight in 5mL of YEPD.
  2. Subclone 1:100 into a flask containing the desired amount of media (50 mL - 2L).
  3. Grow until mid-log phase.
  4. Transfer media to a 50mL conical vial for smaller cultures (100mL or less) or into a 500mL centrifuge bottle for larger cultures.
  5. Spin down at 4k RPM and 4°C for 3-5 minutes.
  6. Discard supernatant and refill with any remaining media. Spin down again and continue until all of the cell culture has been pelleted.
  7. Wash.
    • Use 1x volume of PBS for TAP purification or CoIP.
    • Use 1x volume of TBS for ChIP.
    • Use 1x volume of PBS for RNA extraction. Note that, for RNA extraction, all steps for harvest must be performed at room temperature, including any centrifugation.
  8. Discard the wash supernatant and resuspend the cell pellet in any remaining PBS. It may be necessary to add a small amount of additional PBS.
  9. Transfer the resuspended cell pellet into a 2 mL FastPrep screw-top tube, or into a 50 mL conical vial. Do not overtighten caps.
  10. Spin down at 4k RPM and 4°C for 3-5 minutes for 50 mL conicals or flash spin for 2 mL FastPrep tubes. Discard any remaining supernatant.
  11. Immerse in liquid nitrogen until frozen.
  12. Transfer to -80°C Freezer.