RESUMEN
Large DNA molecules (>20 kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.
RESUMEN
While Bacterial Artificial Chromosomes libraries were once a key resource for the genomic community, they have been obviated, for sequencing purposes, by long-read technologies. Such libraries may now serve as a valuable resource for manipulating and assembling large genomic constructs. To enhance accessibility and comparison, we have developed a BAC restriction map database. Using information from the National Center for Biotechnology Information's cloneDB FTP site, we constructed a database containing the restriction maps for both uniquely placed and insert-sequenced BACs from 11 libraries covering the recognition sequences of the available restriction enzymes. Along with the database, we generated a set of Python functions to reconstruct the database and more easily access the information within. This data is valuable for researchers simply using BACs, as well as those working with larger sections of the genome in terms of synthetic genes, large-scale editing, and mapping.
RESUMEN
Electrostatic forces greatly affect the overall dynamics and diffusional activities of suspended charged particles in crowded environments. Accordingly, the concentration of counter- or co-ions in a fluid-''the salt"-determines the range, strength, and order of electrostatic interactions between particles. This environment fosters engineering routes for controlling directed assembly of particles at both the micro- and nanoscale. Here, we analyzed two computational modeling schemes that considered salt within suspensions of charged particles, or polyelectrolytes: discrete and continuum. Electrostatic interactions were included through a Green's function formalism, where the confined fundamental solution for Poisson's equation is resolved by the general geometry Ewald-like method. For the discrete model, the salt was considered as regularized point-charges with a specific valence and size, while concentration fields were defined for each ionic species for the continuum model. These considerations were evolved using Brownian dynamics of the suspended charged particles and the discrete salt ions, while a convection-diffusion transport equation, including the Nernst-Planck diffusion mechanism, accounted for the dynamics of the concentration fields. The salt/particle models were considered as suspensions under slit-confinement conditions for creating crowded "macro-ions", where density distributions and radial distribution functions were used to compare and differentiate computational models. Importantly, our analysis shows that disparate length scales or increased system size presented by the salt and suspended particles are best dealt with using concentration fields to model the ions. These findings were then validated by novel simulations of a semipermeable polyelectrolyte membrane, at the mesoscale, from which ionic channels emerged and enable ion conduction.
