Orange g powder

A gel shift DNA binding assay used two complementary 50-mer oligonucleotides labeled at the 5′-end with either Cy3 or Cy5. The indicated concentration (5 or 3.6 μM) of Redβ or LiRecT in PBS (or cryo-EM buffer defined below) was mixed with 25 μM (nt) of the indicated oligonucleotide and incubated at 37 °C for 15 min. For some samples as indicated on the gel (lanes labeled “35”, “ad” or “nc”), a second oligonucleotide was added and incubated for an additional 15 min at 37 °C. For all samples the total reaction volume was 30 μl. For visualization 17.5 μl of each complex was mixed with 7.5 μl Orange G dye (65% w/v sucrose, 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.3% Orange G powder from Sigma Life Sciences), loaded onto a 0.8% agarose gel and electrophoresed in 1× TBE at room temperature for 72 min at 96 V. Gels were imaged using a Sapphire Biomolecular Imager (Azure Biosystems) with Sapphire Capture Software (version 1.12.0921.0). Scanning parameters for Fig. 8 were pixel size 100 μm, scan speed high, 2.38 mm focus, intensity 2 for Cy5, intensity 4 for Cy3, black lighting 50, white 37186, gamma 1.37. Scanning parameters for Supplementary Fig. 1a, b were intensity 1 for Cy5, intensity 2 for Cy3, black lighting 50, white 15362, gamma 0.88.