Hindiii digested λ dna

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.

For SSB–DNA samples, 1 nM DNA and 1 nM Alexa dye were mixed with a range of concentrations of SSB. All samples were prepared at 100 μL volume at room temperature (20–25 °C) at the same time prior to beginning SML-FSHS. For plug–plug samples, a sample of 1 nM DNA and 1 nM Alexa was mixed separately from various concentrations of diluted SSB and injected in series. Unless otherwise indicated, 40 mM EB buffer was used as the sample and running buffer for all samples and separations.
For DNA condensation samples, DNA staining was performed in deionized water. Lambda DNA (New England Biolabs) and HindIII Digested Lambda DNA (New England Biolabs) were stained with TOTO-3 iodide (ThermoFisher Scientific) at 10 ng/μL of DNA and 1 μM of dye. The mixtures were allowed to equilibrate in the dark for at least 1 h prior to further analysis. The stained DNA was further diluted to 5 ng/μL in 20 mM EB buffer (free-coiled) or 20 mM EB buffer + 100 μM SPD (condensed) for SML-FSHS analysis. The same staining protocol was followed for HindIII digested λ DNA (New England Biolabs).