Rapid Genomic DNA Extraction and PCR Amplification

DNA extracts were prepared from cultures grown for 24 h at 30 °C. Thus, 100 µL was transferred to a 96-well PCR plate (Applied Biosystems). After centrifugation at 3434× g for 15 min at 4 °C, pelleted cells were boiled (5 min, 95 °C) in 20 µL lysis buffer (0.25% SDS, 50 mM NaOH). Ultra-pure water (180 µL) was added and the supernatant collected after centrifugation. PCR reactions were carried out with Taq DNA Polymerase 2.0 x Master Mix Red (Ampliqon, Ampliqon Denmark) in a final volume 12.5 µL and 1 µL of each DNA extract. Primers are described in Table S1. PCR conditions were denaturation at 95°C for 4 min; 30 cycles at 95°C for 30 s, 50°C for 30 s and 72°C for 2 min; and a final extension step of 72°C for 7 min. Sanger sequencing of IS905::celB PCR product was carried at Macrogen.