Pulsed field gel electrophoresis

C. tropicalis strain MYA-3404 was grown until exponential phase (~2×10⁷ cells/ml). Cells were washed with 50 mM EDTA and counted with a hemocytometer. Approximately 6×10⁸ cells were used for the preparation of 1 ml genomic DNA plugs. The plugs were made according to the instruction manual protocol (BioRad, Cat No. 170–3593) with cleancut agarose (0.6%) and the lyticase enzyme provided by the kit. A 0.6% pulsed field certified agarose gel was prepared using 0.5X TBE buffer (0.1 M Tris, 0.09 M boric acid, 0.01 M EDTA, pH 8) and the PFGE was performed on a CHEF-DR II (Bio-Rad) for 72 h (24 h at 4.5 V/cm/106° with an initial and final switch times 200 s; 48 h at 3 V/cm/106° with an initial and final switch time 700 s). The gel was stained with ethidium bromide (EtBr) and analyzed by using the Quantity One software (Bio-Rad).