Pfge certified agarose

A single plug for each sample was transferred to a 1.5 ml tube and equilibrated for 20 min at room temperature in 1× New England Biolabs (NEB) buffer-2 supplemented with 1× bovine serum albumen (BSA) buffer (New England Biolabs, MA, USA). The buffer was removed and replaced with 200 μl of 1× NEB buffer-2/1× BSA containing 400 units of XbaI restriction endonuclease (New England Biolabs, Ipswich, MA, USA). Digests were performed overnight at 37°C. A 1.0% agarose gel was prepared in 0.5× Tris-borate-EDTA (TBE) buffer (50 mM Tris-Cl pH8.3, 50 mM boric acid, 1 mM EDTA) using PFGE-certified agarose (Bio-Rad Laboratories, Hercules, CA, USA). Samples were separated in 0.5× TBE at 14°C using a Bio-Rad CHEF Mapper XA System (Bio-Rad Laboratories, Hercules, CA, USA) set to resolve DNA fragments of 100–400 kb. Upon run completion, the gel was transferred to a solution of 1 μg/ml ethidium bromide in ultrapure water for 30 min at room temperature, before destaining with two 15-min washes with ultrapure water. Images were captured and the migration distance of the molecular weight markers was measured in millimeters. Molecular weight markers include MidRange PFG Marker I and II (New England Biolabs, MA, USA), and Lambda HindIII marker (Life Technologies, Grand Island, NY, USA).