Fla 5100 imager fluorescence analyzer

Strains carrying plasmids with GAL::RECQL5, GAL::RECQL5-ID, or the empty vector (pYES) were grown selective in SC medium with 2% raffinose to mid-log phase and then 2% galactose was added to induce transcription from the GAL1 promoter. Samples were taken at different times and RNA extraction was performed by acid phenol following standard procedures. Northern blot of kinetics was carried out as previously described (Chávez and Aguilera 1997 (link)). RNA was separated by agarose electrophoresis in a gel containing MOPS 1 × and 2% formaldehyde and transferred to Hybond-N nitrocellulose membranes (GE Healthcare). GAL1 and GAL10 probes were amplified by PCR using primers detailed in Supplementary Table 1. Probes were ³²P-labelled using Klenow polymerase (Roche) and ³²P-dCTP. Signal was acquired using a FLA-5100 Imager Fluorescence Analyzer (Fujifilm) and quantified with MultiGauge 2.0 analysis software (Science Lab). Signal was measured as arbitrarily units (a.u.) and normalized to control condition.

RNA was separated by agarose electrophoresis in a gel containing MOPS 1× and 2% formaldehyde and transferred to Hybond-N nitrocellulose membranes (GE Healthcare). LYS2 and SCR1 probes were amplified by PCR using primers ‘LYS2 probe A fw’ and ‘rv’ or ‘SCR1 .483 rv’ and ‘SCR1 .99 dw’, respectively, purified using Macherey-Nagel's DNA extraction kit and ³²P-labelled using Klenow polymerase (Roche) and ³²P-dCTP. Signal was acquired using a FLA-5100 Imager Fluorescence Analyzer (Fujifilm) and quantified with MultiGauge 2.0 analysis software (Science Lab). Signal was measured as arbitrarily units (a.u.) and normalized to control condition.