Single-stranded DNA curtains were assembled in microfabricated flowcells according to published protocols (37–40 (link)). Briefly, the template and primer oligonucleotides were annealed by heating to 75°C and cooling at a rate of –1°C min–1. Annealed circles were ligated with DNA Ligase (NEB, M0202) for 5 h at room temperature. Long ssDNA molecules were generated in 1× phi29 reaction buffer (NEB, M0269S), 500 μM dCTP and dTTP (NEB, N0446S), 0.2 mg ml–1 BSA(NEB, B9000S), 10 nM annealed circles, and 100 nM phi29 DNA polymerase. The mixture was mixed by pipetting and immediately injected on the flowcell and incubated at 30°C for 20–40 min. All microscope experiments were conducted at 37°C. Images were collected on an inverted Nikon Ti-E microscope in a prism TIRF configuration running NIS Elements (AR 4.30.02). Flowcells were illuminated with 488 and 637 nm lasers (Coherent OBIS) split with a 638 nm dichroic mirror (Chroma). Two-color images were recorded by twin electron-multiplying charge-coupled device (EMCCD) cameras (Andor iXon DU897). Uncompressed TIFF stacks were exported from NIS Elements and further analyzed in FIJI (41 (link)). Data analysis was performed in MatLab R2019a (MathWorks).
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