Seakem gold agarose

PFGE was performed using the CHEF MAPPER (Bio-Rad Laboratories) according to the National Institute of Health guidelines. Briefly, bacterial colonies grown in tryptic soy agar (TSA) (Becton Dickinson and Company) were resuspended in Tris-EDTA buffer (100 mM Tris, 100 mM EDTA) at 20% transparency. The resuspended cells were mixed with 1.2% seakem gold agarose (Lonza, Switzerland) and solidified in a plug mold. The agarose plugs were transferred to cell lysis buffer (50 mM Tris, 50 mM EDTA, 1% sodium lauroyl sarcosine) and lysed by 20 mg/mL proteinase K (Invitrogen, USA) at 55°C with shaking at 100 rpm for 1 h. The lysed plugs were transferred to a plug wash buffer (10 mM Tris, 1 mM EDTA) and washed five times at 55°C with shaking at 100 rpm for 20 min. The washed plugs were digested by the 40 unit of XbaI (Roche, Switzerland) at 37°C for 4 h. Finally, the plugs were separated on 1% seakem gold agarose gel by PFGE in the chamber containing 0.5 × tris-borate-EDTA buffer plus 1.5 M thiourea (Sigma-Aldrich, USA) at 14°C. The PFGE conditions were 6.0 V for 18 h with initial and final switch times of 2.16 sec and 63.8 sec, respectively. The results were analyzed using the Bionumerics software ver. 5.0 (Applied Maths, Belgium). The dendrogram was generated using the Dice coefficient and UPGMA algorithm (0.5% optimization, 1.0% position tolerance).