Charge switch pro plasmid miniprep kit

For PCR amplification of the genes of interest, DNA isolated with ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen, Waltham, MA, USA) was used as template. Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for amplification according to the manufacturer’s instructions; the primers are listed in Table S2. The gel-purified PCR products (GeneJET Gel Extraction Kit, Thermo Scientific, Waltham, MA, USA) and plasmid pSCrhaB2 [27 (link)] were double-digested with NdeI and HindIII (New England Biolabs). After ligation (Quick Ligation Kit, New England Biolabs), the mixtures were transformed into Library Efficiency DH5α™ Competent Cells (Invitrogen) and transformants were selected on LB plates supplemented with trimethoprim (50 µg mL⁻¹). Plasmids were extracted using ChargeSwitch-Pro Plasmid Miniprep Kit (Invitrogen) and the correct insert size was confirmed following NdeI/HindIII double-digestion by gel electrophoresis. Plasmids were introduced into B. cenocepacia J2315 by triparental mating as described [27 (link)]; transconjugants were selected on LB plates supplemented with trimethoprim (200 µg mL⁻¹).

As further validation, several templates (SAG 34-1b, SAG 34-1h, SAG 34-1m, SAG 34-1f, SAG 44.96, SAG 49.94, and HP036) were selected to serve as the basis for cloning and sequencing of the ITS2 gene. Amplicons targeting the transcribed spacer were generated by PCR [35 ] and then purified using illustra™ GFX™ PCR DNA and Gel Band Purification Kits (GE Healthcare, Buckinghamshire, UK) for use in cloning reactions. Cloning was carried out using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). Ligation conditions followed the manufacturer's protocol as optimized for high transformation with minimal ligation time (5 min). All reactions were conducted at room temperature. In order to increase reproducibility of the results, plasmid DNA was isolated from positive colonies using the ChargeSwitch-Pro Plasmid MiniPrep kit (Invitrogen, Carlsbad, CA, USA).