Nolimits dna

Conical glass nanopores were fabricated according to previously reported experimental protocols with a final diameter estimated at 14 ± 3 nm^{16 (link)}. Experiments were performed in 4 M LiCl, 50 mM NaCl, 1 mM MgCl₂ electrolyte buffered at pH 8 with 10 mM Tris-HCl. Individual DNA lengths were chromatography-purified NoLimits DNA (ThermoFisher Scientific). The DNA ruler was synthesised by cutting circular 7249 base m13mp18 ssDNA (New England Biolabs) using the enzymes EcoRI and BamHI to form a linear ssDNA chain 7228 bases in length. The cut scaffold is then purified and mixed in a 1:5 ratio with 212 oligonucleotides which form a double-strand with six equidistant zones of dumbbell hairpins. The mixture is annealed in 10 mM MgCl₂ for 50 min before purification of the excess oligonucleotides using 100 kDa cut-off Amicon filters. All ionic current measurements were recorded using an Axopatch 200B amplifier with an external 8-pole Bessel filter at 50 kHz filtering frequency and then recorded at 250 kHz. For ping-pong experiments where an individual DNA molecule was threaded through the nanopore multiple times by reversing the voltage, a custom Labview 2013 (National Instruments) program was written whereby the voltage was switched 40 ms after the current deviated from a set threshold.