Yeast Chromosome Preparation and Analysis by PFGE

Yeast chromosomes were prepared from 1 mL of yeast cells pre-grown in YP-20 g/L glucose medium and collected at the stationary phase of growth. Cells were treated with Zymolyase 20T and proteinase K in low-melting-point agarose blocks, transferred to a 1% agarose gel, and pulsed-field gel electrophoresis (PFGE) was performed at 8 °C using a Gene Navigator system (Amersham Pharmacia Biotech do Brasil Ltd.a., São Paulo, SP, Brazil) as previously described [29 (link),40 (link),41 (link),44 (link)]. Following electrophoresis, the gel was stained with ethidium bromide and photographed (Gel Doc™ XR, BioRad Laboratories, Hercules, CA, USA). The chromosomes separated by PFGE were transferred to a nylon membrane (Hybond-N⁺, GE Healthcare, Barueri, SP, Brazil) by capillary blotting. Labeling of DNA probes (see below), pre-hybridization, hybridization, stringency washes, and chemiluminescent signal generation and detection were performed with an AlkPhos kit (GE Healthcare) as recommended by the manufacturer. After hybridization, an autoradiography film (Hiperfilm™ ECL, Kodak, GE Healthcare) was exposed to the membrane for 1 to 2 h before it was developed. Images were obtained with Image Lab Software (Gel Doc™ XR) and annotated with Microsoft PowerPoint. Probes were generated by PCR using DNA from strain S288C as template with primers (Table 2) SUC100-F and SUC1320-R for the SUC2 gene.