Genomic DNA extraction
LYSE
The phenol-chlorophorm method was used to extract the bacterial DNA from the samples. Briefly, bacteria were resuspended in Tris-HCl 50 mM pH 8.0, 0.25 M sucrose and 25 mg/ml lysozyme and incubated for 1 h at 37°C. Afterwards, a tungsten carbide bead in sample disruptor (Tissue Lyser, Qiagen, USA) followed addiction of SDS 0.1% was used to lyse the bacteria. The solution was centrifuged, and the aqueous layer was aspirated into a new tube with an equal volume of phenol, which was mixed and centrifuged for 2 min at the highest speed. Again, a new tube was used to transfer the aqueous phase, which was mixed with an equal volume of Tris-saturated phenol/chloroform/isoamyl alcohol (25:24:1). After centrifugating it, a new tube was used to mix the aqueous layer from the previous tube and an equal volume of chloroform, which was centrifugated at the highest speed for 2 min at 4°. Sodium acetate 0.3 M and 2 volumes of ethanol were added to the aqueous phase retrieved from the last centrifugation, which was incubated overnight at −20 °C. One day after, the genomic DNA was precipitated with ethanol 70% and resuspended in water.