Quantity one 1 d analysis system

PCR amplicons and digestion products were resolved by agarose gel electrophoresis with 1.4 % (w/v) agarose in a Tris-acetate-EDTA buffer (TAE). The gels were stained with ethidium bromide (0.5 μg/ml) and visualized under UV light (GelDoc, Biorad, Hercules, USA). The protein samples were separated using a stacking gel containing 4 % acrylamide and 10 % resolving gel by the method of Laemmli [28 (link)]. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. At least two replicates of each experiment were performed with little variation (only one example is shown). The results were analyzed using the Quantity One 1-D Analysis System (Biorad, Hercules, USA). Genetic distance between the isolates was calculated using the Dice coefficient of similarity, and then the strains were clustered in a dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) analysis (data not shown).

Electrophoretic separations of DNA were conducted in gels prepared from agarose (EURx, Gdansk, Poland), Tris-acetate-EDTA buffer (TAE) and ethidium bromide (0.5 µg/mL), using Electrophoresis Sub-Cell and Wide Mini-Sub Cell GT systems (Biorad). The PCR products were visualized under UV light (Gel Doc XR+ gel documentation system, Bio-Rad) and the length of DNA molecules was evaluated against a DNA molecular marker—GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific), O’GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific) and Perfect Plus Molecular Weight Quantitative DNA Ladder (EURx). The results were analyzed using Image Lab Software (Biorad), Quantity One 1-D Analysis System (Biorad) and PyElph 1.4 [60 (link)].