DNA content

  1. When you are ready to harvest the cells, sonicate briefly for 10 sec.
  2. Transfer ~106 cells (about 1 ml log culture) in a 1.5 ml microfuge tube.
  3. Centrifuge at 13,000 g for 30 sec.
  4. Aspirate the supernatant, carefully, without touching the cell pellet.
  5. Add 1 ml of 70% ethanol.
  6. Vortex for 15 sec.
  7. Sonicate for 10 sec.
  8. Store the sample at 4 C for at least overnight. The cells can stay in the fixing solution for up to 10-15 days.
  9. Centrifuge the fixed samples at 13,000 g for 4 min.
  10. Remove supernatant. DON'T TOUCH THE CELL PELLET. IT IS EASY TO LOOSE THE CELLS AT THIS POINT. It is better to leave the last 50 ul of the ethanol solution than trying to remove all of it and lose the cells.
  11. Resuspend the cells in 1 ml of RNase/SYTOX solution, always made fresh.
  12. Sonicate for 15 sec.
  13. Leave at 4 C overnight, in the dark. Use the dark plastic boxes to store the rack with your tubes.
  14. The next day the samples are ready for analysis. Sonicate for 15 sec.
  15. Transfer the samples to 5 ml culture tubes without caps.
  16. Cover the tube rack with aluminum foil, and take them to the flow cytometry facility.

RECIPE for [10X (500 mM) citrate (pH 7.0)] solution:

Dissolve the following in 800 ml distilled water.

      1. 1.71 g of Citric Acid (MW=192.12)
      • 144.42 g of Sodium Citrate (MW=294.10)

    1. You don't need to adjust the pH (if you measured the right amounts the pH will be 7.0).
    2. Adjust volume to 1L with additional distilled H2O.
    3. Sterilize by autoclaving.

RECIPE for the [RNase/SYTOX solution]:

  1. Calculate the total volume you will need (1 ml X #samples). The ingredients below are for 50 ml, enough for 50 samples.
  2. In a 50 ml tube, add the following, in the order shown:
  • 5 ml of [10X Sodium Citrate (pH 7.0)] solution.
  • Add ~25-30 ml of distilled water.
  • Add 360 ul of RNase stock solution [34 mg/ml, Sigma Cat#; R4642-1G].
  • Mix well.
  • Add 2.5 ul of SYTOX stock solution [Invitrogen Cat#: S7020].
  • Bring the volume up to 50 ml with distilled water.
  • Mix well.

The final concentration of the solution will be: 50 mM citrate buffer (pH 7.0), 0.25 mg/ml RNase, 250 nM SYTOX).