Hindered diffusion in agarose gels: test of effective medium model.

The diffusivities of uncharged macromolecules in gels (D) are typically lower than in free solution (D infinity), because of a combination of hydrodynamic and steric factors. To examine these factors, we measured D and D infinity for dilute solutions of several fluorescein-labeled macromolecules, using an image-based fluorescence recovery after photobleaching technique. Test macromolecules with Stokes-Einstein radii (rs) of 2.1-6.2 nm, including three globular proteins (bovine serum albumin, ovalbumin, lactalbumin) and four narrow fractions of Ficoll, were studied in agarose gels with agarose volume fractions (phi) of 0.038-0.073. The gels were characterized by measuring the hydraulic permeability of supported agarose membranes, allowing calculation of the Darcy permeability (kappa) for each gel sample. It was found that kappa, which is a measure of the intrinsic hydraulic conductance of the gel, decreased by an order of magnitude as phi was increased over the range indicated. The diffusivity ratio D/D infinity, which varied from 0.20 to 0.63, decreased with increases in rs or phi. Thus as expected, diffusional hindrances were the most severe for large macromolecules and/or relatively concentrated gels. According to a recently proposed theory for hindered diffusion through fibrous media, the diffusivity ratio is given by the product of a hydrodynamic factor (F) and a steric factor (S). The functional form is D/D infinity = F(rs/k1/2) S(f), where f = [(rs+rf)/rf]2 phi and rf is the fiber radius. Values of D/D infinity calculated from this effective medium theory, without use of adjustable parameters, were in much better agreement with the measured values than were predictions based on other approaches. The strengths and limitations of the effective medium theory for predicting diffusivities in gels are discussed.     

Brownian diffusion and electrophoretic transport of double‐stranded DNA in agarose gels

The field free diffusion constant and the electric field dependence of the electrophoretic mobility and molecular orientation of DNA samples from 5 to 164 kilobase pairs in agarose gels from 0.5 to 2% have been measured by fluorescence recovery after photobleaching and birefringence. In conditions where the reptation predictions hold for the field free diffusion, they partially fail for the DNA size dependence of the low field limit of the electrophoretic mobility. The linear field dependencies of the electrophoretic mobility and orientation factor seem to favor the biased reptation model with fluctuations over the standard biased reptation model, which predicts a quadratic field dependence. The quantitative analysis of the molecular parameters shows, however, that most experiments have been carried out at values of the field where the difference between the two models may be less conclusive. The pore size dependence of the different quantities has been given a particular attention and the role of the distribution of pore sizes in the departures from the reptation predictions is discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 45–59, 1999     

Electron microscopy of beaded agarose gels

Electron microscopy has been carried out on sections of beaded agarose with a wide range of thicknesses, and the results have been analyzed by means of stereological theory using computer graphics. The results agree with a randomly orientated system in which, for 4% gels, the mean molecular weight per unit length of the fiber system is 110 kg mol^?1 nm^?1, and the number average interjunction length is 37 nm, with an asymmetrical distribution resembling a Maxwell-Boltzmann distribution. The spatial distribution of the structure is not uniformly random and there seem to be mi microvoids.     

Purification of DNA from agarose gels

Summary We describe a rapid and easily reproducible modification of the freeze-squeeze method of separating DNA from agarose gels. Our method involves slicing out the agarose gel portion which contains the DNA of interest, freezing this gel slice at –20°C, then centrifuging the frozen slice in a filtration unit which contains a cellulose acetate filter. The agarose is retained on the filter and the filtrate contains the DNA. DNA purified in this manner could be completely digested with restriction endonucleases and completely ligated with DNA ligase, without further purification. The percentages of recovery for various sizes of linear and plasmid double-stranded DNA ranged from 57 to 69%. The procedure takes less than 30 minutes to perform.     

Determination of hematocrit based on diffusion of an inert molecular probe from agarose gels into whole blood

Whole blood hematocrit was determined by an approach which depends on the diffusion of an inert probe, to which red blood cells are impermeable, from a small agarose gel into a stirred, much larger blood sample. Blood cells influence the diffusion rate of the probe by, on the average, physically blocking a fraction of the gel surface. The blocking effect increases with the hematocrit. Cyanocobalamin (B-12) was found to be a suitable probe because it did not penetrate, bind to, or lyse blood cells and was not bound by plasma solutes. The loss of B-12 from gels in contact with blood was monitored by determination of the absorbance change at 540 nm of gels which had been quickly rinsed. The visible spectrum of B-12 in agarose gels was identical to the spectrum in water. Beer's Law was obeyed in 1-mm thick agarose gels over a concentration range of 0.1-0.8 mM. Based on the results from 48 blood samples covering the hematocrit range 25-69, a least-squares line was generated with a slope, -3.46 X 10(-3) delta A/hematocrit unit, a Y intercept of 0.295, and a correlation coefficient of 0.971. The precision of the technique was +/- 9.7%. The assay was insensitive to mean corpuscular volume and sample volume as long as the latter was 50-fold larger than the gel volume. The diffusion coefficient for B-12 in 1% agarose gels was found to be 1.4 +/- 0.2 X 10(-6) cm2 sec-1.     

Mesoscopic gels at low agarose concentration: perturbation effects of ethanol.

Aqueous agarose solutions at low concentrations (0.5 g/liter) were temperature quenched below the spinodal line to form mutually disconnected mesoscopic gels. In the presence of 6% ethanol, these solutions, obtained by quenching at the same temperature depth as in pure water, appear much more fluid, as determined by probe diffusion experiments. We show by static and dynamic light scattering that this can be explained by the solvent-mediated effects of ethanol, leading to a globular shape of mesoscopic agarose gels, rather than to an extended rodlike structure observed in pure water. Our findings show the significant effects of solvent perturbations on particle condensation and, therefore, may be useful in understanding the role of the solvent in the folding of biomolecules.     

Simple DNA elution from agarose gels

We developed a simple DNA elution method from agarose gels. After electrophoresis of DNA in an agarose gel, the DNA fragment to be recorved was excised out of gel with a scalpel. The excised gel was placed in the middle of small Parafilm piece, and the Parafilm was folded over the gel piece. Using the petriplate, or thumb, the gel piece was pressed between the Parafilm. Upon squeezing, the DNA inside of the gel gets extruded along with the buffer. The droplets were collected with a pipet. The DNA was then purified by conventional phenol: chloroform extraction method. Typical yields are greater than 50% as determined by UV absorbance.     

The structure of agarose gels,and the accommodation within them of irregularly shaped particles

This paper shows how the number of cross-linkage points (nodes) in a random reticulum, such as an ideal polysaccharide gel, may be calculated in accordance with mathematical principles. The influence of nodal configurations upon the statistical geometry of the reticulum is discussed, and it is shown from experimental evidence that the nodal configurations in agarose gel are nonrandom. A method is given for calculating the accommodation probability of an irregularly shaped particle in a reticulum, which is relevant to the theory of gel chromatography and to the distribution of cells in tissues permeating a network of capillaries or veins.     

Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.     

Recovery of DNA segments from agarose gels

After electrophoresis, DNA can be efficiently recovered by solubilization of agarose gels with NaClO₄, followed by retention of DNA on glass fiber filters. After removal of the NaClO₄ by ethanol, the DNA can be extracted with a low salt buffer.     

Extraction of nucleic acids from agarose gels

A rapid, simple and versatile method is described for the extraction from agarose gels of small plasmid molecules and DNA fragments generated by restriction endonucleases. The method may be used also for the extraction of RNA from agarose-urea gels. It is based on the partitioning of nucleic acid molecules into 1-butanol as their quaternary ammonium salts, leaving the neutral agarose in the aqueous phase. The nucleic acid is then recovered as the sodium salt by partition back into an aqueous phase. Nucleic acid samples were found to be unaffected by the treatment, as judged by their ability to be ligated, transformed, nick-translated, and used in an in vitro protein-synthesizing system.     

Orientation of DNA in agarose gels.

An orientation of the lambda DNA during the electrophoresis in agarose gels was measured by a microscopic linear dichroism technique. The method involved staining the DNA with the dye ethidium bromide and measuring under the microscope the polarization properties of the fluorescence field around the electrophoretic band containing the nucleic acid. It was first established that the fluorescence properties of the ethidium bromide-DNA complex were the same in agarose gel and in a solution. Then the linear dichroism method was used to measure the dichroism of the absorption dipole of EB dye bound to lambda DNA. In a typical experiment the orientation of two-tenth of a picogram (2 x 10(-13)g) of DNA was measured. When the electric field was turned on, the dichroism developed rapidly and assumed a steady state value which increased with the strength of the field and with the size of DNA. A linear dichroism equation related the measured dichroism of fluorescence to the mean orientation of the absorption dipole of ethidium bromide and to an extent to which the orientation of this dipole deviated from the mean. The observed development of dichroism in the presence of an electric field was interpreted as an alignment of DNA along the direction of the field. The increase in the steady state value of dichroism with the rise in the strength of the field and with the increase of the size of DNA was interpreted as a better alignment of DNA along the direction of the field and as a smaller deviation from its mean orientation.     

Electrophoresis of DNA in oriented agarose gels

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.     

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.     

Mapping of zein polypeptides after isoelectric focusing on agarose gels

Isoelectric focusing of zein in agarose gels gives sharp separations of at least 25 bands noted among 25 corn-belt inbreds. Six inbreds provided standard bands which were used to construct a pattern map. A method is provided for comparing bands, identified by distance from the cathode, which differ only slightly in position. The 25 inbreds were separated into five groups on the basis of pattern similarity. Some groups contained inbreds derived from widely different sources. Zein isoelectric focusing in agarose should be useful for genotype identification and for determination of varietal purity.     

Purification and cloning of DNA fragments fractionated on agarose gels

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.