Optiquant cyclone plus machine

Resection of DSB ends was analyzed at an HO endonuclease-induced DSB at the MAT locus on chromosome III using Southern blots as previously described (7 (link),11 (link)). Galactose induction, sample collection, DNA isolation and purification were carried out as described by Chen et al. (11 (link)). Purified DNA was digested with EcoRI and separated on a 0.8% agarose gel followed by transferring onto a nylon membrane. Radiolabeling of DNA probes was performed following manufacturer's instruction (Takara). Southern blotting and hybridization with radiolabeled DNA probes was performed as described previously (7 (link),11 (link)). The blot was exposed in a Phosphor screen. Signal on the screen was captured by scanning in an OptiQuant Cyclone Plus machine (Perkin Elmer). Intensities of target bands were analyzed with OptiQuant software (Perkin Elmer) and normalized to the TRA1 probe. Resection rate was calculated as previously described (7 (link)). Three independent experiments were performed for each strain.

To examine the repair kinetics of ectopic recombination, we cultured yeast cells in the pre-induction medium (YEP–Raffinose) overnight to the early log phase. Then, 2% of galactose was added to induce the HO cut that generates a single DSB on chromosome V. Samples were collected at different time points. Genomic DNA was extracted using a standard phenol extraction method and digested with EcoRI. Purified DNA was resolved on 0.8% agarose gel followed by transfer onto a positively charged nylon membrane (Perkin Elmer). Southern blotting and hybridization with radiolabeled DNA probes were performed as described previously (Zhu et al., 2008 (link); Chen et al., 2012 (link)). The blot was exposed in a phosphor screen. The signal on the screen was captured by scanning in an OptiQuant Cyclone Plus machine (Perkin Elmer). We quantified and normalized the pixel intensity of target bands to that of corresponding parental bands on blots. The resulting values were further normalized to that of the control sample (uncut). Three independent experiments were performed for each strain.

Yeast cells were grown overnight in the pre-induction YEP–raffinose medium (1% yeast extract, 2% peptone, and 2% raffinose) to a density of ~1 × 10⁷ cells/ml. DSB induction was initiated by adding 2% galactose. For testing the resection under replication stress, 200 mM HU (final concentration) was added to each sample when starting the galactose induction. Samples were collected at different time points following break induction. DNA isolated by glass bead disruption using a standard phenol extraction method was digested with EcoRI and separated on 0.8% agarose gels. The resolved DNA was transferred onto a Nylon hybridization transfer membrane (Perkin Elmer). Radiolabeling of DNA probes was carried out according to the manufacturer's instructions (Takara). Southern blotting and hybridization were carried out as described previously (Chen et al., 2012 (link)). The signal on the phosphor screen was captured by scanning with an OptiQuant Cyclone Plus machine (Perkin Elmer). Quantities of DNA loaded on gels for each time point were normalized using the TRA1 DNA probe. The resulting values were further normalized to that of the control sample (uncut). Three independent experiments were performed for each strain.