Midori green dna

The amplified products from all types of the PCR reactions were resolved on a 2% or 0.8% agarose gel (Sigma-Aldrich, USA) and visualized with Midori Green DNA (Nippon Genetics, Germany) under UV light using a Gel Doc camera system (Bio-Rad, USA) and analyzed with Quantity One software (Bio-Rad, USA). PCR assays were repeated twice to confirm the correctness of the assignment of the investigated strains to their respective patterns.

All PCRs reactions were conducted in a DNA Thermal Cycler T100™ (Bio-Rad, Dublin, Ireland). Amplified products were resolved on a 2% agarose gel (Sigma-Aldrich, Wien, Switzerland) and visualized using Midori Green DNA (Nippon Genetics, Dueren, Germany). Band patterns were visualized under UV light and photographed using a Gel Doc camera system (Bio-Rad) and analyzed with Quantity One software (Bio-Rad). For each PCR reaction, 1 µl of sterile water added to the PCR mixture used as a negative control in the experiment. PCR assays were performed twice to ensure that the strains were correctly assigned to their respective patterns.
For the purpose of the next step of the phylogenetic analyses (RAE-PFGE analysis and MLST), eleven of the tested E. coli strains were selected: UPEC 15279.21, 1119 and 15035.8; APEC 1288, 1239, 189A and 60C and RepEC 200E, 209E, 212E and 305C. These strains were selected based on the obtained results of phylogroup assignment and virotyping.

For strains that had reduced susceptibility to vancomycin (MIC = 1–2 mg/L), the vanA and vanB genes were searched. Genomic DNA was isolated using the Genomic Micro AX Staphylococcus Gravity kit (A&A Biotechnology, Poland) according to the manufacturer’s protocol. The genes were detected using the previously described primers and parameters [25 (link), 26 (link)]. DNA amplification was performed in a thermocycler (Biometra, Germany). The reaction products were identified by electrophoresis (70 V, 1.5 h) in 1% (w/v) agarose gels containing the dye Midori Green DNA (Nippon Genetics Europe, Germany). The sizes of the amplification products were identified by using the DraMix Marker or the DNA Marker 2 (A&A Biotechnology, Poland). Enterococcus faecalis NCTC 12201 (vanA) and E. faecalis ATCC 51299 (vanB) were used as controls.