PFGE Protocol for Separation of High-Molecular-Weight DNA

PFGE was performed as previously described [25 (link)] with modifications enabling greater separation of high-molecular-weight DNA molecules, such as chromosome XII, from the rDNA cloud. Plugs were placed in the wells of a 0.8% agarose gel (D5 agarose (Conda, Torrejon de Ardoz, Madrid, Spain) in 1x TAE) and sealed with the same agarose. Until stated otherwise, electrophoresis was performed for 24 hours in 1x TAE buffer at 6 V/cm, 12°C, ramping 0.8, angle 120°, switch time 60–85 s, using a CHEF Mapper® XA Pulsed Field Electrophoresis System (BioRad, Hercules, CA, USA). After electrophoresis, DNA was stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich) for 30 min, washed twice with water for 15 min, and photographed using a 302-nm UV light for DNA visualization with a charge-coupled device camera (Fluorchem Q Multi Image III, Alpha Innotech, San Leandro, CA, USA).