The University of Iowa Carver College of Medicine

Flow Cytometry Facility

DNA Content of Yeast

The following protocol is for DNA content of yeast using propidium iodide.

Reagents

  • EtOH
  • 50mM NaCitrate pH 7.5 (IMPORTANT: stock should be filtered before storing and before subsequent use with cells)
  • Propidium iodide (1.6 mg/ml)
  • RNase A (0.25mg/ml)
  • Proteinase K (20mg/ml)

Staining

  1. Add 10ml absolute EtOH to 5ml of a sample for at least 1 hour in a 15ml conical centrifuge tube (cell concentration should be 0.5 OD at 600nm in spectrophotometer or approximately 15 x 106/ml.
  2. Spin down samples at 5500 rpm (5,450 x g) for 5 minutes at 5°C.
  3. Remove supernatant.
  4. Turn 15ml conical centrifuge tubes upside down for 15 minutes to let residual EtOH drain into cap.
  5. Add 1ml NaCitrate to pellet and leave for 30 minutes at room temperature (cells will resuspend over time)
  6. Resuspend and transfer to 1.5ml Eppendorf tubes.
  7. Spin at 10,000 rpm for 1 minute at 5°C.
  8. Remove supernatant.
  9. Resuspend in 1ml NaCitrate with 0.25mg/ml RNase A.
  10. Incubate at 55°C for 1 hour.
  11. Add 10µl of 20mg/ml Proteinase K.
  12. Incubate at 55°C for 1 hour.
  13. Add 10µl of 1.6mg/ml propidium iodide.
  14. Store at 4°C overnight in the dark.
  15. Transfer to tubes that fit flow cytometer
  16. Vortex and sonicate samples to disrupt aggregates

This protocol is based upon the procedure in "A Checkpoint Regulates the Rate of Progression through S Phase in S. cerevisiae in Response to DNA Damage," by Paulovich and Hartwell, 1995.