Quantifying DNA Recombination Events

DSBs, dHJs, crossovers, and non-crossovers were analyzed by DNA physical assays in combination with Southern blot as described previously (39–41 (link)). Briefly, the genomic DNA was extracted from synchronized yeast cells at desired time points in SPM and digested with Xho I. One-dimension (1D) gel electrophoresis was carried out, followed by a Southern blot to detect DSBs, crossovers, and non-crossovers. Two-dimension (2D) gel electrophoresis in combination with Southern blot was performed to detect IH-dHJs and IS-dHJs. For both crossovers and non-crossovers analysis, the genomic DNA was digested with Xho I and NgoM IV, separated by 1D gel electrophoresis, and detected by Southern blot. The probe for the Southern blot was labeled with α-³²P-dCTP by a random labeling system (Thermo Fisher, Cat#18187-013). All DNA species in Southern blot were visualized using a phosphorimager (the Cyclone Plus Storage Phosphor System, PerkinElmer) and quantified using Quantity One software (Bio-rad).