Toto 3 dye

EB-HCl buffer consisted of Bis-Tris, EACA (ε-aminocaproic acid), and HCl (hydrochloric acid) at the constant ratio (1× EB-HCl = 2 mM EACA + 8 mM Bis-Tris + 1.8 mM HCl, pH 7). EACA and Bis-Tris (Sigma-Aldrich) were dissolved in deionized water at 500 mM as the stock concentration to prepare the EB-HCl buffer with stock hydrochloric acid at 6 M (Sigma-Aldrich). EB-HCl buffers with concentration ranging from 1×, 2.5×, 5×, to 10× were used as the high-salt buffer. The low-salt buffer was ultrapure water (simplicity water purification system, Millipore). Hind III digested λ DNA (New England Biolabs) was used as the DNA sample for all experiments. In order to eliminate an additional fragment (27 kbp) formed by the annealing of the cohesive ends of fragments 23 and 4.4 kbp, the DNA sample was heated at 60 °C for 3 min, followed by cooling on ice for 3 min. Then, the DNA at 5 ng/μL was stained with 1 μM TOTO-3 dye (ThermoFisher Scientific) in the EB-HCl buffer. The DNA sample was stored on ice in a dark environment for at least 1 h and was further diluted to a final concentration of 5 ng/mL in the EB-HCl buffer before each run. Alexa 647-NHS ester (ThermoFisher Scientific) stocked at 1 μM was spiked in the blank EB-HCl buffer at 1 nM and used as a “free dye” marker for flow velocity calibration.