Fish sperm dna

Oligonucleotides used for EMSA are listed in Supplementary Table 6. For AP1, AP1m1, AP1m2, AGI-II and S-AGI, complementary single-stranded oligonucleotides were annealed in annealing buffer (10 mM Tris pH 7.5, 150 mM NaCl and 1 mM EDTA). The resulting double-stranded DNA with a protruding G was fluorescently labelled by end filling: 4 pmol of double-stranded DNA was incubated with 1 unit of Klenow fragment polymerase (Ozyme) and 8 pmol Cy3-dCTP or Cy5-dCTP (GE Healthcare) in Klenow buffer during 2 h at 37 °C, followed by 10 min enzyme inactivation at 65 °C. Binding reactions were performed in 20 μl binding buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% glycerol, 0.25 mM EDTA, 2 mM MgCl₂, 0,01% Tween-20 and 3 mM TCEP) with 10 nM labelled probe, 1 × (28 ng ml⁻¹) fish sperm DNA (Roche) as nonspecific competitor and 25–500 nM proteins.
Competition assays were performed in duplicates and 1–100 × fish sperm DNA (Roche) was used in the binding reaction. Signal quantification was performed in using ImageLab v2.0.1 (Bio-Rad Laboratories). Signal of each protein–DNA complex was quantified relatively to total DNA signal. For Fig. 4e, each binding reaction was performed in triplicate. Uncropped gels are presented as Supplementary Fig. 10.

The flow cell consisted of a square glass capillary tube (1 × 1 mm² section, 5 cm long, VitroCom, Mountain Lakes, NJ). For each measurement, 250 μl of DNA and magnetic beads suspension were injected, in the absence of a magnetic field, into the capillary tube previously functionalized as follows. First, a solution of 100 mg/ml polystyrene (average MW230000, Sigma Aldrich) in toluene was injected into the capillary. Then, the capillary was drained and dried with compressed air. In this way, the internal walls were uniformly coated with polystyrene (31 (link)). Next, 5 μg of sheep polyclonal anti-digoxigenin antibody (Roche) in 100 ml with 10 mM PBS were incubated in the capillary for 2 h at 37°C. Unbound anti-digoxigenin was eliminated by rinsing the capillary tube with PBS-Tween-20. The functionalized surface was passivated for 2 h at 37°C with a solution consisting of 10 mM PBS at pH 8 supplemented with 0.1% Tween-20, 1 mg/ml fish sperm DNA (Roche) and 3 mM NaN₃ (32 (link)). Finally, the capillary tube was rinsed with PBS-Tween-20 and incubated for 1h to allow the DNA to bind to the lower capillary tube surface. For storage, several capillaries could be prepared simultaneously and kept at 20°C after air drying them.