Gel doc it photo documentation system

Genomic restriction was performed according to the standardised PulseNet Salmonella protocol [30 (link)]. Agarose-embedded DNA was digested with 40 U of SpeI for 3 h at 37 °C. The restriction fragments were separated by electrophoresis in Tris-borate-EDTA (44.5 mM Tris-borate, 1 mM EDTA; pH 8.0) at 14 °C using a CHEF-DRIII (Bio-Rad, Milan, Italy). Electrophoresis conditions were as follows: (Block I) initial switch time 2 s, final switch time 10 s, voltage 6 V, included angle 120°, and run time 13 h; (block II) initial switch time 20 s, final switch time 25 s, voltage 6 V, included angle 120°, and run time 6 h. The size standard used for all gels was XbaI-digested DNA from Salmonella Braenderup strain, the universal size standard used by all PulseNet laboratories. The PFGE agarose gels were stained with ethidium bromide (40 µg/mL) and the DNA band images were acquired by the Gel Doc-It photo documentation system (Gel Doc-It photo documentation system, UVP, Upland, CA, USA). A dendrogram of pulsotype relationships was developed through the unweighted pair group method using arithmetic averages (UPGMA) with BioNumerics software version 6.5 (Applied Maths). Pulsotypes were assigned to the same clusters if they exhibited 80% similarity in the dendrogram.

Genomic restrictions were performed as previously reported [13 (link)] and according to the standard operating procedure for PulseNet PFGE of Escherichia coli O157:H7, E. coli non-O157 (STEC), Salmonella serotypes, Shigella sonnei, and Shigella flexneri [14 (link)]. Agarose-embedded DNA was digested with 40 U XbaI for 4 h at 37 °C. The fragments were separated in 1% agarose gel (Pulsed Field Certified Agarose; BioRad, Milan, Italy) in Tris–borate–EDTA (44.5 mM Tris–borate, 1 mM EDTA; pH 8.0) at 14 °C, using a CHEF-DRIII (Bio-Rad, Milan, Italy) apparatus. Electrophoresis conditions were as follows: initial switch time 7.3 s, final switch time 24 s, voltage 6 V, included angle 120°, and run time 19 h. XbaI-digested DNA fragments from the Salmonella Braenderup strain H9812 were used in each gel as universal size standards. The agarose gels were stained with ethidium bromide (40 g/mL) and the DNA band images were acquired by the Gel Doc-It photo documentation system (UVP, Upland, CA, USA).