Chromosomal DNA Separation by PFGE

Preparation of chromosomal DNAs and their separation by PFGE were performed as previously described^{26 (link)}. Chromosomal DNAs were prepared in 0.8% low melting agarose gels (Nacalai tesque, 01161-12) and resolved using a CHEF-DRII pulsed-field electrophoresis system (Bio-Rad) under the following conditions. For broad-range PFGE, a 1600 s pulse time at 2 V cm⁻¹ for 42 h followed by a 180 s pulse time at 2.4 V cm⁻¹ for 4 h, at 4 °C in 1× TAE buffer using 0.55% Certified Megabase agarose gel (Bio-Rad, 161-3109)). For short-range PFGE, a 40–70 s pulse time at 4.2 V cm⁻¹ for 24 h, at 4 °C in 0.5× TBE buffer using 0.55% Certified Megabase agarose gel. DNAs were stained with 0.2 µg mL⁻¹ EtBr (Nacalai Tesque, 14631-94) and detected using a Typhoon FLA9000 gel imaging scanner (GE Healthcare). Gel images were processed using ImageJ software or Adobe Photoshop Elements (Adobe, San Jose, CA).