Positively charged nylon membrane

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.

Total RNA was extracted and separated on 15% urea-PAGE gel, followed by transferred to positively charged nylon membrane (PerkinElmer, Waltham, MA, USA) using Trans-Blot SD Semi-Dry transfer Cell (Bio-Rad, Hercules, CA, USA). Oligonucleotide probes used for hybridization with miRNA were labeled with at their 5′ end. U6 was used as the loading control. The sequence of the probe was: miR-26a: 5′-ATTC AAGTTTTGAAACAGGTGTA-3′.