Orange gel loading dye

Other than the restriction endonuclease protection assays (REPA) performed as part of a round of REPSA, REPAs were also performed with defined DNAs to ascertain their protein-DNA binding characteristics. Defined DNA REPAs contained 1 nM each 63-bp IRD7-labeled SbtR consensus DNA (red) and 86-bp IR8-labeled REPSAis control DNA (green) in a 5 μl reaction volume together with 1× NEB CutSmart buffer and the indicated final concentration of SbtR. These were incubated at 55°C for 20 min to affect binding, followed by equilibration at 37°C for 5 min. IISRE (0.2 U BpmI) was then added and cleavage allowed to proceed at 37°C for 5 min before stopping on ice. NEB Orange Gel Loading Dye supplemented with 1% SDS (2 μl) was added and the samples analyzed by native 10% PAGE analysis as described above. Visualization of IR-dye labeled DNA species was performed using a LI-COR Odyssey Imager.

Target (57 bp, 1 pmol) and off-target (40 bp, 1 pmol) duplexes were treated with increasing concentrations of Pt(ii)-TFO hybrid (2.5–50 eq.) in triplex buffer (10 mM phosphate, 150 mM NaCl, 2 mM MgCl₂ pH 6–7) and incubated at 37 °C for 48 h. Orange gel loading dye (1 μL, 6X, NEB) was added and the sample solution was loaded onto a 20% PAGE gel (50 mM Tris acetate, 150 mM NaCl, 2 mM MgCl₂, pH 6–7). Electrophoresis was performed at 70 V for 240 min in triplex running buffer (50 mM Tris acetate, pH 6–7). Polyacrylamide gels with TO-containing sequences were visualised and imaged on a Syngene G:Box Mini 9 gel documentation system. Subsequently, TO-containing sequences were then post-stained with SYBR Gold and visualised similarly. Gels with non-TO-containing sequences were post-stained with SYBR Gold and visualised as before.