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Nolimits dna

Manufactured by Thermo Fisher Scientific

NoLimits DNA is a high-performance DNA cloning and amplification solution. It provides reliable and consistent results for a variety of molecular biology applications.

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3 protocols using nolimits dna

1

DNA Ruler Fabrication and Nanopore Manipulation

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Conical glass nanopores were fabricated according to previously reported experimental protocols with a final diameter estimated at 14 ± 3 nm16 (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.
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2

DNA Translocation in Nanopore Sensing

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The DNA translocation experiments were performed by flowing a nanopore buffer containing 1 nM of 5 kbps NoLimits DNA
(ThermoFisher Scientific, Waltham, MA) through the microfluidic chip. Specifically, the 10 mM Tris-EDTA buffer containing DNA was
sent to the nanopore cis chamber with minimal to no dilution in volume. During sensing, the cischamber contained 10mM Tris-EDTA and the trans chamber contained 1M KCl. The salt concentration in both nanopore
chambers were not equal, resulting in microscale mixing near the pore’s vicinity. Such a salt gradient is sufficient to
perform the experiment as previously described.[49 (link)]
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3

Investigating DNA Size Effects

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To investigate the effect of biomolecule size, individual DNA fragments with 25 bp, 100 bp, 500 bp, 1000 bp, 10 000 bp, 15 000 bp and 20 000 bp were purchased from Thermo Fisher Scientific Inc. under the brand of NoLimits DNA. For each DNA size, the stock vial consisted of 10 mg of DNA at a concentration of 0.5 mg ml À1 in 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. To visualize and quantify the DNA uptake, DNA molecules were stained in the stock vials using YOYO-1 dye (1 mM in DMSO from Thermo Fisher Scientific Inc.). The bp : YOYO-1 dye molecule staining ratio was 10 : 1 to achieve a sufficient fluorescence signalto-noise ratio, without significantly influencing the contour length of the DNA molecules. 15, 16 Staining was carried out on ice for a period of 1 hour.
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