Smai endonuclease

The clonal diversity of Lactococcus lactis was determined by PFGE analysis of the strains after phenotypic sorting. Bacterial cultures and agarose plugs were prepared, as described previously by Lortal et al. [24 (link)]. The plugs were equilibrated for one hour in a restriction buffer (CutSmart, New England Biolabs, Beverly, MA, USA) at 4 $°$ C, and were then transferred to a fresh digestion buffer containing 15 units SmaI endonuclease (New England Biolabs, Beverly, MA, USA) for one hour. The plugs were then incubated at 25 $°$ C for 4 h. PFGE was performed in a Bio-Rad CHEF DRII electrophoretic cell on 1% (w/v) agarose gel (Ultrapur, Gibco-BRL, Inchinnan, Scotland) in 0.5x TBE buffer (45mM Tris, 45mM boric acid, 1mM EDTA, pH 8.0) at 200 V and 14 $°$ C, under the following conditions: initial time—2 s, final time—25 s, total running time—21 h. Strain CIRM-BIA 127 was used as a home-made ladder for this study. After migration, the gels were stained with gelRed, visualized using UV light and then analyzed with GelCompar software (BioNumerics, Applied Math, Austin, TX, USA). Conversion, normalization, and further analysis were performed using the Pearson coefficient and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis with BioNumerics software (Applied Math, Sint-Martens-Latem, Belgium).

PFGE analysis of the S. aureus isolates was performed according to a standardized protocol [90 (link)] with the SmaI endonuclease (New England Biolabs, Beverly, MA, USA) using a CHEF-DR III apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA). DNA from Salmonella enterica serotype Braenderup H9812, which was digested with XbaI, was used as a reference size standard, while PFGE patterns were analyzed using the FPQuest software (Bio-Rad Laboratories Inc. Pty Ltd.). PFGE profiles were compared using the Dice correlation coefficient with a maximum position tolerance of 1.5% and an optimization of 1.5%, while the Unweighted Pair Group Method using Averages (UPGMA) was used for clustering analysis and the generation of a dendrogram.