Diploid cells were grown to saturation overnight in YC media lacking adenine and methionine. Cells were used to inoculate YEPD +/- 20 µg/ml doxycycline cultures, which were incubated with shaking, 30°C for 5 hours. Cells were counted and used to inoculate 40 ml YEPD +1 µM estradiol +/- 20 µg/ml doxycycline cultures at 2×104 cells/ml and incubated with shaking, 30°C for 95 hours. Samples were harvested and plated as described above for the LOH assay. To shift FOB1 expression from repression to activation, 15 ml of the + doxycycline culture was harvested at the 65 hour time point, washed, and resuspended in 15 ml fresh YEPD +1 µM estradiol.
