Determining Chromosomal Insertions in Gmm

To determine the number of chromosomal insertions, genomic DNA from tetracycline-treated Gmm females and normal Gmm individuals were restricted with HindIII endonuclease, electrophoresed on 1% agarose gel in 1× TBE buffer, and transferred to a positively charged nylon membrane according to Southern protocol [68] (link). The membrane was hybridized at 55°C with 350 ng of a 569 bp probe corresponding to part of the wsp gene labeled with the Gene Images Alkphos Direct labeling system (GE Healthcare, Little Chalfont, UK) using the random primer method following manufacturer protocols. Signal detection was performed using CDP-star followed by exposure to autoradiographic film (X-OMAT AR, Kodak). The absence of cytWol from the tetracycline-treated Gmm DNA was confirmed by a PCR assay, which resulted in only a single 16S rRNA amplification product originating from the chromosomal insertions [45] (link).