Chef dr 2 electrophoresis system

The CeuI fragments were separated by a CHEF-DR II electrophoresis system (Bio-Rad). Electrophoresis was performed on a 1% agarose (Seakem Gold agarose, BioWhittaker Molecular Applications) gel and 0.5X TBE buffer (Bio-Rad) at 11°C. The electrophoresis conditions were divided into two stages to separate the DNA fragments of various sizes: First stage, pulse time ramped from 6.75 s to 2 min for 20 h at 4 V cm^-1 and in a second stage, pulse time ramped from 0.22 to 5.10 s for 15 h at 6 V cm^-1.
The agarose gels were radiated with UV light for 1 min in a UV Crosslinker (UVP) to fix the DNA. The gels were washed in 250 mM HCl solution for 15 min with moderate shaking. Next, the gels were washed in denaturing buffer (1.5 M NaCl, 0.5 M NaOH) for 2 h and later washed in a neutralization buffer (0.5 M Tris/HCl, 1.5 M NaCl; pH 8.0) for 2 h. The DNA fragments were then transferred onto N+nylon membrane (Amersham Biosciences) via Southern blotting as described previously (Sambrook et al., 1989 ). Finally, the membrane was radiated with UV light to fix the DNA (1 min; UV Crosslinker, UVP).

The genetic relationship among the OXA-23-producing A. baumannii strains was determined with PFGE using the restriction enzyme ApaI (New England BioLabs, Ipswich, MA, USA). The restriction fragments were separated on a 1% (w/v) agarose gel in a 0.5% tris-borate-EDTA buffer in a CHEF-DR II electrophoresis system (Bio-Rad Laboratories, Richmond, CA, USA) for 19 h at 14°C, using a pulse ramp rate changing from 5s to 60s, at 6 V/cm.The restriction patterns were analysed using BioNumerics software v. 6.0 (Applied Maths, Sint-Martens-Latem, Belgium). Percentage similarity between fingerprints was scored using the Dice coefficient[20 ]. Theunweighted pair group method with arithmetic mean method was used to construct the dendrogramwith a 1.5% tolerance limit.