Certified megabase agarose

DNA plugs for pulse-field gel electrophoresis were prepared using CHEF Genomic DNA Plug Kits (Bio-Rad Laboratories Inc., Hercules, CA, USA) as follows. DOA9 was cultured for 6 d at 28°C in peptone salts yeast extract medium [16 (link)]. Bacterial cells were collected by centrifugation, washed twice with 0.85% NaCl, and resuspended with 0.85% NaCl to an OD₆₀₀ of 5. The cell suspension (0.5 mL) was centrifuged at 8,000 × g for 5 min at 10°C, then resuspended with 0.5 mL of cell suspension buffer and mixed thoroughly with 0.5 mL of 2% CleanCut agarose (Bio-Rad Laboratories Inc., Hercules, CA, USA). The mixture was transferred to plug molds and solidified at 4°C for 30 min. The solidified agarose plugs were treated with lysozyme (1 mg/mL) at 37°C for 4 h and with proteinase K (30 U/mL) at 50°C overnight. Fragments of 225–6,000 kb and 225–2,200 kb were separated on 0.8% certified megabase agarose (Bio-Rad Laboratories Inc., Hercules, CA, USA) in TAE buffer or 1% certified megabase agarose in 0.5xTBE buffer, respectively. Contour-clamped homogeneous electric field (CHEF) electrophoresis was conducted at 14°C in a temperature-controlled cooling unit using the autoalgorithm mode in the CHEF Mapper gel electrophoresis system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gel was stained with 0.5 mg mL⁻¹ ethidium bromide for 1 h and then destained in H₂O for 1 h.

A 30 ml aliquot of mid-exponential culture (about 2 × 10⁸ cells) was taken for each sample and processed using the CHEF Yeast Genomic DNA Plug Kit (Bio-Rad, 170-3593), according to the manufacturer’s instructions (6 × 10⁸ cells per ml of agarose plug). A 3 mm slice of each plug was loaded on a 0.8% agarose (Certified Megabase Agarose, Bio-Rad, 161-3108) gel made in 1× Tris-Borate-EDTA buffer (TBE). Chromosomal DNA was separated using a CHEF-DR II system (Bio-Rad) with 1× TBE as running buffer, at 14°C for 90 hr at 3V/cm, with switch times ramping from 300 to 900 s. The gel was stained with 1 μg/ml ethidium bromide and photographed, before transfer of DNA to Hybond-XL membranes (GE Healthcare Life Sciences, RPN 203 S), using a VacuGene XL vacuum blotting system (GE Healthcare Life Sciences). Membranes were hybridized with a probe for the rDNA (Chromosome XII, 466875-467891), labeled with [α-32P]-dCTP using a Random Primed DNA labeling Kit (Roche, 11 004 760 001). For detection, membranes were exposed to BAS Imaging Plates (Fujifilm), which were then analyzed using a FLA-5100 scanner and AIDA Image Analysis software (Raytest).