Phage lambda pfge ladder

S1-PFGE was conducted to determine the size of large plasmids. Briefly, agarose-embedded DNA was digested with S1 nuclease (New England BioLab) at 37°C for 1 h. The restriction fragments were separated by electrophoresis in 0.5 Tris-borate-EDTA buffer at 14°C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA, USA) with pulse times of 2.16 to 63.8 s. Phage Lambda PFGE ladder (New England BioLab) was used as DNA size marker. The gels were stained with GelRed, and DNA bands were visualized with UV transillumination (Bio-Rad). Chromosomal and plasmid DNA of S. typhimurium strains were transferred and cross-linked onto nylon membrane and hybridized with a DIG-labeled oqxAB probe using DIG High Prime DNA Labeling and Detection Starter Kit I (Roche) following manufacturer’s instructions to determine the localization of oqxAB and aac(6′)-Ib-cr genes in S. typhimurium genetic materials.

Clonal relationship between representative Salmonella isolates was examined by pulsed-field gel electrophoresis (PFGE) according to the PulseNet PFGE protocol for Salmonella36 (link). S1-PFGE was conducted to determine the size of large plasmids. Briefly, agarose-embedded DNA was digested with S1 nuclease (New England Bio-Lab) at 37 °C for 1 hr. The restriction fragments were separated by electrophoresis in 0.5 Tris-borate-EDTA buffer at 14 °C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA) with pulse times of 2.16 to 63.8 S. Phage Lambda PFGE ladder (New England Biolab) was used as DNA size marker. The gels were stained with GelRed, and DNA bands were visualized with UV transillumination (Bio-Rad). Southern blot hybridization was carried out by following the manufacturer’s instructions of the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics), using the different PMQR gene and bla_CTX-M-64 digoxigenin-labeled probes.