‫בס"ד‬
FACS PROTOCOL – SACCHAROMYCES CEREVISIAE
1. Harvest cells
2. Centrifuge samples for 10 minutes.
Carefully and thoroughly aspirate supernatant.
3. Resuspend and "wash" cells with 1ml ddH20.
Vortex cells.
4. Centrifuge cells (1 min).
Aspirate supernatant.
 Resuspend each sample in 1ml 70% Ethanol.
5. Leave cells overnight in ethanol solution in -20°.
6. Centrifuge samples for 10 minutes.
Carefully and thoroughly aspirate supernatant.
Resuspend cells with 1ml Na-Citrate (50mM, pH7).
7. Sonicate cells: [Setting- 30%].
10 seconds -- 1 second ON, one second OFF.
8. Centrifuge cells (1 min).
Carefully aspirate supernatant.
9. Resuspend cells with 300µl Na-Citrate(50mM, pH7) + 7.5μg RNase, per sample (0.25mg/ml)
Incubate for 2 hours at 37°.
10. ADD 15µl Proteinase K (20mg/ml) to each sample.
Incubate for 2 hours at 37°.
11. Centrifuge cells (1 min).
Carefully aspirate supernatant.
12. Resuspend cells with 400µl Na-Citrate(50mM, pH7) + PI (16µg/ml).
Vortex Cells.
Cover samples and keep away from light sources.
Incubate for 30 minutes at room temperature and store in 4° (for a week maximum).
13. Transfer samples to FACS tubes.
Vortex Cells.
Keep away from light sources.
To get a good reading, the FACS machine needs about 1x106 cells in the final solution, although it
reads only 20,000. Keep in mind that you have the right amount of cells and ≥400μl of solution at the
final stage.