Preparation of yeast cells for FACS analysis
A Dohlman Lab Protocol
1. Grow cells overnight to saturation.
2. Dilute to A600nm=0.2 in the same media, grow to A600nm=1.0.
3. Sonicate 1.5 ml for 10 sec, to disperse cells.
4. Centrifuge 10 sec at maximum speed in a tabletop microfuge, and resuspend the pellet in 2 ml 70% EtOH, incubate overnight at 23 C with gentle shaking.
5. Centrifuge as before, wash twice with 1 ml of 50 mM Tris-HCl pH 7.8, resuspend in 0.8 ml 50 mM Tris-HCl with 200 ug heat inactivated RNAseA, incubate at 37 C overnight.
6. Centrifuge cells and resuspend in 0.5 ml of 50 mM Tris-HCl pH 7.8, 2.5 mg pepsin (powder) and incubate at 37 C for 30 min.
7. Centrifuge and wash once with 1 ml 200 mM Tris-HCl pH 7.5, 211 mM NaCl, 78 mM MgCl2, and resuspend in 0.55 ml of the same buffer containing 50 ug propidium iodide (100X stock in water).
Li, F., Flanary, P. L., Altieri, D. C., and Dohlman, H. G., Cell division regulation by Bir1, a member of the IAP (inhibitor-of-apoptosis) family in yeast. J. Biol. Chem. 275:6707-6711, 2000.