Dynabead myone streptavidin t1

Microchambers were prepared similarly to what has been previously described (25 (link),39 ). In brief, a microchamber with ∼30 μl volume was created between two glass coverslips (Fisherbrand, Thermo Fisher Scientific, Waltham, MA, USA) separated by a parafilm gasket with a narrow inlet and outlet to reduce evaporation of the reaction buffer, and a wide, central observation area (40 (link)). DNA tethers were attached through a digoxigenin at one end to the chamber bottom passivated with anti-digoxigenin (Roche Life Science, Indianapolis, IN, USA) and at the other end to either a 320-nm-diameter streptavidin-coated polystyrene bead (Spherotech, Lake Forest, IL, USA) for TPM experiments or a 1.0-μm–diameter streptavidin-coated paramagnetic bead (Dynabead MyOne Streptavidin T1, Invitrogen, Life Technologies, Grand Island, NY, USA) for magnetic tweezing. The chamber was filled with a 10 mM Tris–HCl (pH 7.4), 200 mM KCl, 0.5 mg/ml α-casein buffer and stored in a sealed box to maintain high humidity at 4°C. Before use the chamber was flushed with 200 μl of λ buffer (10 mM Tris–HCl (pH 7.4), 200 mM KCl, 5% DMSO, 0.1 mM EDTA, and 0.1 mg/ml α-casein).

Chambers were prepared as previously reported (16 (link),20 (link),21 (link)). In brief, micro-chambers with an approximate volume of ∼30 μl were prepared by laser-cutting a gasket from parafilm and mildly heating to seal it between two coverslips (Fisherbrand, Thermo Fisher Scientific, Waltham, MA, USA). The channel inlet and outlet were narrow to reduce the evaporation of buffer during observation (Supplementary Figure S2). The digoxigenin-labeled ends of DNA molecules were attached to the glass surface coated with polyclonal anti-digoxigenin (Roche Life Science, Indianapolis, IN, USA) and the opposite biotin-labeled ends to 1.0 μm-diameter, streptavidin-coated paramagnetic beads (Dynabead MyOne Streptavidin T1, Invitrogen, Grand Island, NY, USA). The chamber surfaces were passivated with 0.1 mg/ml BSA to prevent non-specific sticking of the DNA and beads to the surface. Prepared chambers were filled with buffer (10 mM Tris–HCl pH = 7.4, 200 mM KCl, 0.5 mg/ml α-casein) and stored in a high humidity box at 4°C up to 24 h or used directly after gently flushing with 200 μl λ buffer (10 mM Tris–HCl pH 7.4, 200 mM KCl, 5% DMSO, 0.1 mM EDTA, 0.2 mM DTT and 0.2 mg/ml α-casein).