DNA Staining and Imaging in Nanofluidics

For nanofluidics experiments, DNA was stained with YOYO-1 (YOYO, Invitrogen) in a molar ratio of 1:5 to the total number of basepairs in the sample, and with netropsin (Sigma-Aldrich) in a molar ratio of 150:1 with respect to YOYO. The samples were initially mixed in 5x TBE (Tris-Borate-EDTA, Medicago, diluted with ultrapure water from 10x tablets) and left to equilibrate at room temperature for about 20 minutes. As an example, 2 μL of plasmid DNA (100 μM, bp) was mixed with 2 μL λ-DNA (100 μM, bp), 2 μL of YOYO-1 (40 μM) and 3 μL of netropsin (4000 μM). 5x TBE was then added in order to obtain a final volume of 10 μL. Subsequently, the samples were diluted to 0.05x TBE with ultrapure water to a final concentration of typically 0.4 μM of DNA (bp). The mixing in high ionic strength was performed to enable rapid equilibration of YOYO on DNA35 (link). Beta-mercaptoethanol (BME, Sigma-Aldrich) was added in 2% (v/v) to suppress excessive photonicking of the plasmids. λ-DNA (48502 bp, New England Biolabs) was used as standard for measurements of the sizes of the plasmids and was measured in the same conditions as for the plasmids.