![]() ![]() Gel electrophoresis showed high assembly yield and purity, whereas fluorescence correlation spectroscopy confirmed that the tetrahedrons had a diffusion coefficient (26.7 μm 2 s −1) consistent with the expected size (20 nm). MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. In either case, collected ssDNA-containing fractions were homogeneous and impurity free.įinally, 8.4 μg of a 1000-nt ssDNA fragment was purified and used alongside with site-specific short oligonucleotides (staples) to assemble 63-bp edge length tetrahedrons. Freeze drying is shown to cause bead damage to both resins, but to different extents. In multimodal chromatography, however, the elution pattern was reversed, highlighting the importance played by hydrophobicity. In anion exchange chromatography, the less-charged ssDNA eluted before the dsDNA. To isolate the target ssDNA from dsDNA and other PCR impurities, anion-exchange (Q-ligand) and multimodal chromatography (Capto TM adhere ImpRes) were explored using stepwise gradients with increasing NaCl concentrations. Alternatively, we present a chromatography-based method to purify ssDNA scaffolds from aPCR mixtures, which can be used in the context of DNA-origami techniques.ĪPCR was performed to generate single and double-stranded DNA (dsDNA) from the M13mp18 genome. Each scaffold is usually purified by agarose gel extraction, a technique that is laborious, limited, not scalable, presents low recovery yields and a low-quality product. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500–3500 base, object-specific, single-stranded DNA (ssDNA) scaffolds. ![]()
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