Generating genomic platforms to study Candida albicans pathogenesis

Mélanie Legrand; Sophie Bachellier-Bassi; Keunsook K. Lee; Yogesh Chaudhari; Hélène Tournu; Laurence Arbogast; Hélène Boyer; Murielle Chauvel; Vitor Cabral; Corinne Maufrais; Audrey Nesseir; Irena Maslanka; Emmanuelle Permal; Tristan Rossignol; Louise A. Walker; Ute Zeidler; Sadri Znaidi; Floris Schoeters; Charlotte Majgier; Renaud A. Julien; Laurence Ma; Magali Tichit; Christiane Bouchier; Patrick Van Dijck; Carol A. Munro; Christophe d’Enfert

albicans ORFs in the pDONR207 vector has been described (34) . Briefly, ORFs, ranging from 90 bp to 5,295 bp, were amplified from genomic DNA of C. albicans strain SC5314 in 96-well plates using the Thermo Scientific Phusion High-Fidelity DNA Polymerase and 30 cycles of amplification, with elongation time varying from 1 to 3 min according to the ORF size. After ethanol precipitation, Gateway™-compatible amplified ORFs were recombined into pDONR207 (Invitrogen) using the Gateway™ BP Clonase™ II Enzyme Mix (Invitrogen). Reaction mixes containing pDONR207, the PCR products and the BP Clonase™ were incubated overnight in 96-well plates, at room temperature. After adding proteinase K (Invitrogen) and incubating 10 min at 37°C, the BP reactions were directly used for bacterial transformation. 45-50 µl of chemically competent E. coli DH5α were added to the BP reactions and incubated for 30 min at 4°C. After heat-shock at 42°C for 35 s, 150 µl of SOC medium (20 ml YPD + 2 ml LB + 1 ml Hepes) was added to the transformation reactions and the samples were covered with breathing films and incubated for 1.5 h at 37°C with shaking. Then 100 µl of each sample were plated onto LB agar containing 10 µg/ml Gentamicin and incubated overnight at 37°C. The remainder of the transformation reactions were stored at -80°C in 30% glycerol. A single colony from each transformation reaction was inoculated in 400 µl 2YT + 10 µg/ml Gentamicin in 96 deep-well microplates. After 36hgrowth at 37°C, the cultures were used for plasmid extraction and for -80°C storage.