3915 96 well black plate

In vitro fluorescence assays were carried out in a buffer containing 40 mM HEPES, pH 7.5 and 125 mM KCl. Other conditions, including temperature, concentration of MgCl₂, DFHBI (or DFHBI-1T), ligand (or cell extract) and RNA were varied in different experiments and are indicated in the figures. The RNA was renatured in buffer at 70 °C for 3 min and cooled to ambient temperature for 5 min prior to addition to the reaction solution containing DFHBI (or DFHBI-1T), buffer, and ligand. Binding reactions were performed either in 100 μL (96-well plate) or 30 μL (384-well plate) volumes and were incubated at the indicated temperature in either a Corning Costar 3915 96-well black plate or Greiner 781077 384-well black plate until equilibrium was reached, which typically takes 30 to 60 minutes. The fluorescence emission was measured using a Molecular Devices SpectraMax Paradigm Multi-Mode detection platform plate reader (Sunnyvale, CA) with the following instrument parameters: 448 nm excitation, 506 nm emission for DFHBI or 470 nm excitation, 510 nm emission for DFHBI-1T.

In vitro fluorescence assays were carried out in binding buffer containing 100 nM RNA, 10 µM DFHBI, 40 mM HEPES, pH 7.5, 125 mM KCl, and 3 or 10 mM MgCl₂, as indicated in the figures. Other conditions, including temperature and concentration of ligand, were varied in different experiments as indicated. The RNA was renatured by heating to 72 °C for 3 min in the binding buffer then cooled to ambient temperature for 5 min prior to addition to the reaction solution. DFHBI was added to the solution containing buffer and RNA, and then ligand (or water for no ligand control) was added before fluorescence measurement. Binding reactions were performed in 100 µL volumes and were incubated at the indicated temperature in a Corning Costar 3915 96-well black plate or a Greiner Bio-One 384-well black plate in a Molecular Devices SpectraMax Paradigm Multi-Mode detection platform plate reader (Sunnyvale, CA). The fluorescence emission was measured during 30 to 60 min total with the following instrument parameters: 448 nm excitation, 506 nm emission.