Coating Flow Cells with Biotinylated DNA

The flow cells coated with lipid bilayer with attached biotin (DOPC plus DOPE-biotin [1%]) were prepared essentially as described in Han and Mizuuchi, 2010 (link) and rinsed with a buffer containing 25 mM Tris–HCl pH 7.4, 150 mM NaCl, and 5 mM MgCl₂ and 0.1 mM CaCl₂. Sonicated and biotinylated DNA was prepared as follows: 250 μl of 10 mg/ml sonicated salmon sperm DNA (Sigma) was sonicated for an additional 5 min (Misonix sonicator 3000, output level 6, pulsed on/off for 10 s each at 16°C) to size-weighted average length of ~500 bp. In order to biotinylate the DNA ends, the sonicated DNA (1 mg/ml) was incubated with 40 μM biotin-17-dCTP (Invitrogen) and 0.6 units TdT (NEB) in the buffer specified by the enzyme manufacturer at 37°C for 30 min. The reaction was stopped by heating at 70°C for 10 min, and unincorporated biotin-17-dCTP was removed by using S-200 HR Microspin columns (GE Healthcare). The DNA was ethanol precipitated and resuspended in TE buffer. To coat the flow cell with sonicated DNA, the DNA prepared as above was dissolved to 1 mg/ml in 25 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl_2, and 0.1 mM CaCl₂, infused into the assembled flow cell, and incubated overnight at 4°C. Unbound DNA was removed by rinsing with 50 mM Tris–HCl (pH 7.4), 100 mM KCl, 2 mM MgCl₂, and 10% glycerol.