Yeast Cell Cycle Arrest and DNA Extraction

A 50mL yeast culture in YPD was grown to OD600 of 0.6 and then arrested in G1-phase by addition of alpha factor (50 ng/mL) for 2h. As indicated, cells were treated with auxin at a concentration of 1mM for 30 min at 30°C to degrade AID-tagged Ask1or with nocodazole at a concentration of 15 μg/mL together with 1%DMSO for 2h at 30°C to destabilize microtubules. To release the cells from the arrest, 125U of Pronase (Sigma- Aldrich, 53702-25KU) and potassium phosphate buffer to a final concentration of 20mM was added. If necessary, 200mM HU was added in the release to induce S phase checkpoint activation. Samples for genomic DNA extraction were taken before the release and every 8min after releasing the cells from the arrest by adding 4.5mL of the culture to 500μL of 1% sodium azide solution (w/v) in 0.2M EDTA. The cells were washed once with water (4.000g, 3 min at 4°C) and the resulting yeast pellets were snapfrozen in liquid nitrogen.
For DNA extraction, the cell pellets were resuspended in buffer RINB (50mM Tris-HCl pH8, 0.1M EDTA, 0.1% (v/v) beta mercaptoethanol). Zymolyase was added to a final concentration of 2% (w/v). After incubating for 1h at 37°C, the solution was supplemented with 1% SDS (w/v), 0.2M NaCl, 0.1 mg/mL RNAse A, and 0.2 mg/mL proteinase K. After incubation for 1h at 55°C, DNA was isolated by phenol-chloroform extraction followed by ethanol precipitation. DNA pellets were suspended in 50μL of H₂O. 5–10μg of DNA was then digested with EcoRI. The reactions were diluted 1:10 in H₂O and analyzed by quantitative PCR using primers 0463/0466 (ARS305), 0552/0553 (ARS313), 0970/0971 (ARS315), 0837/0838 (ARS316), and 0834/0835 (ChrVI).