Lambda ladder pfge marker

PFGE tests were performed according to Ochoa et al. [15 (link)]. First, the UPEC O25b strains were embedded in low-melting-point (LMP) agarose blocks from Promega Corporation (Madison, WI, USA). Next, digestion of the samples was performed using 20 U of the restriction enzyme Xba1 (New England Biolabs, Ipswich, MA, USA) at 37 °C for 20 h. The PFGE shift was performed for 24 h at 200 V (7 v/cm) to an angle of 120° at 14 °C, with an initial pulse of 2.16 s and a final pulse of 13.58 s, in a CHEF Mapper system (Bio-Rad Life Science Research, Hercules, CA, USA). The macrorestriction products in the PFGE gels were stained with GelRed^® Nucleic Acid Stain (Biotium, Fremont, CA, USA), visualized with UV light, and digitized using the CCD Camera Documenting System BK04S-3 (Biobase, Mexico City, Mexico). PFGE pulsotypes were analyzed using NTSYSpc v2.02j and PAST v4.03 software, and the clonality degree was estimated according to the criteria described by Tenover et al. [22 (link)]. A lambda ladder PFGE marker (New England Biolabs, Hertfordshire, England, UK) was used as a molecular weight marker in the PFGE assay.

PFGE was performed according to a previously published protocol12 (link)16 (link). Briefly, strains were grown overnight on 5.0% blood agar plates. The cells were harvested and washed two times with solution buffer (Tris-HCl, 0.01 M; EDTA, 0.1 M; pH 8.0). Streptococcus cells were lysed with 1.0 mg/mL lysozyme and 1.0 mg/mL proteinase K (Sigma, USA). The bacterial suspensions were mixed with an equal volume of 1.0% low-melting-point agarose (Cambrex, USA) and pipetted into a 100 μl plug. A solution in CLB (50 mM Tris, 50 mM EDTA, 1% SDS, 0.1 mg/mL proteinase K) was then added. The plugs were incubated in a solution with 12 U of SmaI restriction enzyme (Takara, China) and its associated buffers and then then sent for PFGE assay using the following program: a switch time of 4–40 s, 20 h, a 120° angle and a voltage gradient of 6 V/cm in a CHEF Mapper XA (Bio-Rad, USA). A lambda ladder PFGE marker (New England Biolabs, USA) was used as a DNA size marker. The gels were stained with ethidium bromide and photographed under UV light. PFGE patterns were then analysed and compared using BioNumerics version 6.5 software (Applied Maths BVBA, Belgium).The unweighted-pair group method was used with arithmetic averages and Dice’s coefficient in the UPGMA Programme to process the data.