Optimisation of Candida albicans typing by pulsed-field gel electrophoresis

Six strains of Candida albicans were subjected to pulsed-field gel electrop horesis (PFGE) using the CHEF-DRIII system (BioRad). Hansenula wingei YB-46 62-VIA and Saccharomyces cerevisiae YNN 295 (BioRad) were used as size mark ers (1.05-3.13 and 0.22-2.2 megabase pairs [Mbp] respectively) for comparis on of DNA molecules. The DNAs were resolved by a three-block protocol with pulse times of 120 s for 24 h, 240 s for 36 h and 300 s for 17 h. The volta ge was set at 4.5 V/cm for the first two blocks and 4.0 V/cm for the final block. PFGE was carried out under these conditions using different agarose concentrations, types and concentrations of buffer, temperatures, and sizes of agarose gel plug. The resolution and mobility of DNAs were affected by some of these variables. Separation of C. albicans by PFGE was optimal at 1 2 degrees C with 1.0 x Tris-borate-EDTA (TBE) buffer using 1.2% agarose. Re solution of banding patterns was dependent on size of DNA plug used.