Appendix A. Box fingerprinting.
BOX-PCR was used to evaluate whether bacterial isolates assigned to the same species by sequencing the 16S rRNA gene corresponded to different strains. For that, we used the primer BOX A1R (Rademaker et al. 1998), according to the methodology described by Bathe et al. (2006). Briefly, amplification was performed in 20µl reaction mixtures containing 1µl of bacterial genomic DNA, 1.25mM of each dNTP (Euromedex, Mundolsheim, France), 5µM of the primer, 10% DMSO (Invitrogen, Cergy Pontoise, France), 2.5µg T4 gene 32 protein (Qbiogene, Carlsbad, USA), 6.35mM MgCl2 (Invitrogen), 2µl of PCR buffer (Invitrogen) and 1.2U of Taq polymerase (Invitrogen). The thermocycling conditions consisted of an initial denaturation step at 94°C for 5 min, followed by 25 cycles of 94°C for 45 s, 50°C for 1.5 min, and 65°C for 8 min; with a final extension step at 65°C for 16 min. PCR was carried out in a Biometra thermal cycler (Goettingen, Germany). The BOX-PCR products were examined by electrophoresis in 2% agarose gels (Invitrogen) for 2.5 h at 100 V and visualized by staining with ethidium bromide.
Bathe, S., W. Achouak, A. Hartmann, T. Heulin, M. Schloter, and M. Lebuhn. 2006. Genetic and phenotypic microdiversity of Ochrobactrum spp. FEMS Microbiology Ecology 56:272–280.
Rademaker, J. L. W., F. J. Louws, and F. J. de Bruijn. 1998. Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting. Pages 1–26 in A. D. L. Akkermans, J. D. Van Elsas, and F. J. de Bruijn, editors. Molecular Microbial Ecology Manual. Kluwer, Dordrecht, The Netherlands.