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).

Reference:
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.