Agarose Concentration Research Articles

Dynamics of a Polymer Solution in a Rigid Matrix

Dynamic light scattering measurements of the diffusion coefficient D and the Rayleigh ratio R θ are reported for dextran molecules confined in agarose gel networks of various concentrations. In this condition, the light scattered by the dextran is some 2 orders of magnitude less intense than that from the agarose. Three molecular weights of dextran were investigated: 7 x 10 4 , 5 x 10 5 , and 2 x 10 6 g mol -1 . For the lowest molar mass it is confirmed that, below the dextran overlap concentration c * , the product DR θ is independent of the agarose concentration, showing that the reduction of the rate of diffusion inside the gel is the result of a decrease in the osmotic pressure in the confined geometry. For the higher molar masses, entanglement effects between the dextran and the network become noticeable in the more highly concentrated gels. The dynamic light scattering intensity measurements are also found to yield reasonable estimates of the molar mass M w and radius of gyration R G of the trapped dextran molecules. The second virial coefficient A 2 is positive, indicating that the agarose-water matrix acts as a good solvent for dextran, but the ratio of R G to the hydrodynamic radius is less than 1.5. These results are interpreted in terms of branching of the dextran molecule.

Structure−Properties Relation for Agarose Thermoreversible Gels in Binary Solvents

The structure of agarose gels prepared in aqueous binary solvents has been studied by means of X-ray and neutron small-angle scattering. The scattering curves are interpreted by considering a random assembly of straight fibers displaying cross-section polydispersity. The cross-section polydispersity is well accounted for with a distribution function of the type w(r) ∼ r-λ bounded by two radii, rmin and rmax, and where λ depends upon both the nature of the binary solvent and the agarose concentration. Departure from the Porod regime at wide scattering angles suggests the existence of free and/or dangling chains that do not participate in the network elasticity (loose chains). The fraction of loose chains gradually vanishes upon increasing the agarose concentration. Experimental elastic modulus−concentration relations obtained on the same type of samples point to the occurrence of entropic elasticity once analyzed in light of the existence of loose chains. Entropic elasticity is rather consistent with disor...

Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects.

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.

A Single Polymerase Chain Reaction-Based Protocol for Detecting Normal and Expanded Alleles in Myotonic Dystrophy

The myotonic dystrophy (DM) expansion varies from 50 to 4000 CTG repeats in the 3' untranslated region of the DMPK gene. Direct analysis by Southern blot, after restriction enzyme digestion of genomic DNA, is the method of choice for studying the DM mutation. A long polymerase chain reaction (PCR)-formatted protocol, which involved a single genomic in vitro amplification followed by high concentration agarose gel electrophoresis and oligo-specific hybridization, was used to amplify normal alleles and DM alleles in all examined ranges of expansion (up to 3,700 CTGs) starting from a small amount of genomic DNA (> or = 15 pg). This method is quick, sensitive, and reproducible and reduces the cost of diagnostic laboratory processing.

ＢＭＰ‐アガロース複合体の骨形成能

To evaluate the osteoinductive activity and the biocompatibility of BMP-agarose composite, three kinds of experimental samples (BMP pellet samples, agarose samples, and BMP-agarose composite samples) were implanted into the hindquarter muscle pouch of ddy conventional mice.The mice were killed 21 days after implantation, and the hind limbs were examined by roentgenographic and histologic methods. The osteoinductive activity of the samples was confirmed by computer assisted analysis of soft X-ray photographs to determine the volume of induced new bone.BMP pellet samples induced stable heterotopic new bone formation.Three weight-percent agarose samples induced no bone formation. Histologically, the implant was surrounded by a thin fibrous layer, but did not cause inflammatory cell aggregation. Therefore, agarose has excellent biocompatibility and is very stable in living tissue.BMP-agarose composite samples induced stable heterotopic new bone formation. Histologically the induced bone region was surrounded by a thin fibrous tissue layer and consisted of bone-like calcified tissue with osteocyte-like cells, cartilaginous cells, and bone marrow. However, the bone induced by BMP-agarose composite samples seemed to be less mature than that induced by BMP pellet samples. This suggested that the initial effects of BMP remained because of slow release from the composite, and that the BMP continuously formed bone, which contained less mature tissue during the initial period of bone formation.A comparison of the volume of induced bone showed a trend toward an increase in bone with higher concentrations of agarose in the composite. The BMP-agarose composite, made with 2 and 3 weight-percent agarose solution, induced significantly greater bone formation than did BMP pellet samples.These results show that BMP-agarose composite has the advantage of acting as an osteoinductive implant material in vivo.

Evaluation of agar and agarose gels for studying mechanical impedance in rice roots

Agar and agarose gels were evaluated as systems to mechanically impede roots of rice (Oryza sativa L.). Two-layer gels were used so that seedlings established in a layer of weak gel (0.35% weight/volume) and then grew downwards to encounter a treatment gel of up to 5.0% (w/v). Agarose gels were stronger than agar gels of the same concentration, reaching a maximum penetrometer resistance of 1.2 MPa at a concentration of 5.0%, compared to 0.3 MPa with agar. The 5.0% agar gel stimulated elongation of the seminal axis by 40% in seedlings of variety TN1 (compared with elongation in the 0.2% gel), but decreased it by 15% in the variety Lac 23. Although increasing agarose concentration decreased seminal axis elongation in both varieties, the seminal axis did not reach the lower layer of treatment gel when the concentration of the treatment gel was greater than 2.0%. The decreased root elongation was therefore a non-mechanical inhibition. In experiments conducted using a different batch of agarose, these inhibitory effects were not seen and strong agarose gels stimulated seminal axis elongation. It was concluded that the agar and agarose gel systems studied were unsuitable for studying the effect of mechanical impedance on the elongation of rice roots and that great care should be taken in interpreting the results of experiments using gels as a growth medium.

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmotic stress, created by high agarose or carbohydrate concentrations, on the regeneration of fertile plants from protoplast-derived colonies of several indies (IR43, Jaya, Pusa Basmati 1) and japonica (Taipei 309) rice varieties. Observations of the cultures developed on media containing one of these carbohydrates (cellobiose, fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose), each at 88 mM, indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration. Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety, compared to shoot regeneration from 4-32% of the tissues in sucrose-supplemented medium. Media containing 288 mM maltose or an equimolar combination of 88 mM maltose and 200 mM mannitol, caused water loss from calli and promoted the growth of embryogenic calli. These calli formed shoots with greater frequencies when subsequently transferred to shoot regeneration medium with 88 mM maltose. A medium containing 88 mM maltose and semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarose had a similar beneficial effect on the growth of embryogenic calli and simultaneously supported high-frequency (48-55%) shoot formation. The optimum shoot regeneration frequencies (60-78%) were obtained when protoplast-derived colonies were serially cultured on to shoot regeneration medium containing 1.0% (w/v) agarose for 4 weeks, followed by a 2-week culture period on the same medium with 0.5% (w/v) agarose. Plants regenerated on medium containing maltose and/or 1.0% (w/v) agarose were phenotypically normal and fertile.

Ternary complexes in agarose gels prepared from binary solvents

AbstractThe thermoreversible gelation of agarose has been investigated in four different aqueous binary solvents: Water/dimethyl sulfoxide, water/N,N‐dimethylformamide, water/N‐methylformamide, and water/formamide. The phase diagrams have been subsequently established as a function of agarose concentration and solvent composition. These diagrams suggest the formation of ternary complexes agarose/water/cosolvent.

Transient electric birefringence study of agarose aggregates in water‐dimethyl sulphoxide mixtures

AbstractThe dynamical behavior of agarose aggregates in a mixture of water and dimethyl sulphoxide (DMSO) has been investigated using a Transient Electric Birefringence (TEB) set up. Different agarose concentrations for different water/DMSO proportions were considered. The relaxation of the birefringence curves can be fitted with a stretched exponential of the form Δn(t) = Δn0 exp(‐(Dt)0.5). The exponent 0.5 suggests a high polydispersity of the aggregates. The rotational diffusion coefficient, D, depends strongly on the water/DMSO proportion. For small values of the applied electric field it presents a maximum for a water volume fraction equal to 0.5. A strong dependence of D on the applied electric field is also observed. However, the same kind of behavior as for weak fields is recovered when a subtle representation of the data is used. Differential Scanning Calorimetry (DSC) results also show a maximum in the temperature of fusion of the aggregates for a water volume fraction of 0.5. This set of results suggest strongly that « complexes » of water/DMSO/agarose are formed in the mixture and that the process of aggregate formation is more « efficient » for a water/DMSO mixture of same proportions.

Purification of plasmid DNA by an improved electrophoretic protocol

Plasmid DNA from Escherichia coli was isolated by electroelution carried out in an agarose gel that contains an incorporated dialysis membrane. As the relative mobility of circular plasmid DNA to linear chromosomal DNA increases when the agarose concentration is decreased, we were able to purify plasmids of up to 50 kbp in 0.3% agarose gel in Tris acetate buffer yielding 10-60 μg DNA ml bacterial culture.

Dose distribution of proton beams with NMR measurements of fricke-agarose gels

Fricke-agarose gels have been irradiated with a proton beam. Then samples have been extracted at different depths with respect to the beam penetration distance, corresponding to different irradiation doses. Relaxation times T 1 and T 2, measured at 17 MHz, appear sensitive to this kind of radiation. In particular, T 2 exhibits three components T 2 a, T 2 b and T 2 c, the first two being sensitive to proton irradiation. At 1% agarose concentration, the relaxation rates R 1 = 1 T 1 , R 2 a = 1 T 2 a and R 2 b = 1 T 2 b of samples irradiated with both modulated and unmodulated beams, increase with the dose, irrespective of the beam energy. The yield G of Fe 3+ ions per 100 eV of absorbed energy is always higher than that obtained for gamma irradiated samples.

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.

The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis.

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The β-glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.

Hydraulic permeability of agarose gels

AbstractA new technique was developed to measure the hydraulic permeability of reinforced gel membranes and allows calculation of the Darcy permeability (κ) of the gel. It was applied to agarose with concentrations ranging from 2.0 to 7.3%. To create membranes that would be thin enough to yield easily measured filtration rates at modest applied pressures and yet withstand handling, gels were cast on woven polyester meshes. The resulting membranes had thicknesses of 70–100 μm and a fractional open area of 0.32. To correct for the presence of the mesh, finite‐element solutions were obtained for the pressure field in the 3‐D region occupied by the gel. For the meshes used here, the hydraulic permeability of the reinforced membrane was calculated to be 0.47–0.55 times that for a layer of pure gel, the exact value depending on the thickness of the composite membrane. The principal determinant of κ was the agarose concentration, but there was a secondary effect of applied pressure. The Darcy permeability extrapolated to zero applied pressure (κ0) varied from 616 nm2 for 2.0% agarose to 22 nm2 for 7.3% agarose. At a given gel concentration, the value for κ0 was as much as twice the value for κ measured at the maximum pressure difference of 20 kPa. This method can be adapted to a variety of other gel materials.

Combined chiral crown ether and β-cyclodextrin for the separation ofo-, m-, andp-fluoro-D,L-phenylalanine by capillary gel electrophoresis

A combination of (β-CD) with a chiral crown ether, 18-crown-6-tetracarboxylic acid (18C6TA), has been used for the chiral capillary gel electrophoretic (CGE) separation of the six enantiomers and positional isomers in a mixture ofo-, m-, andp-fluoro-D,L-phenylalanine; the isomers were successfully resolved in 0.3% agarose −2×10−2 mol L−1 18C6TA −2×10−2 mol L−1 β-CD. The method has also been successfully extended to the separation of the enantiomers of 13 other amino acid or primary amine substrates. The role of the chiral crown ether was studied. The effects of the concentrations of 18C6TA, β-CD and agarose, and of pH and applied field strength on the migration time and separation factors of the analytes are reported.

Stimulatory effect of water stress on plant regeneration in aromatic Indica rice varieties

Frequency of regeneration of fertile plants from cell suspensions was significantly increased using water stress treatments in two commercially cultivated Indian aromatic rice varieties, Basmati 385 and Pusa Basmati 1. The water stress treatments included the use of 1.0% (w/v) agarose instead of 0.5% (w/v) for medium solidification, inclusion of mannitol (0.1, 0.2 and 0.4 M) in regeneration medium, or 24 h partial desiccation of calli. When the agarose concentration of the regeneration medium was increased from 0.5% to 1.0% (w/v), the frequency of shoot formation in Pusa Basmati 1 from cell suspension-derived calli increased by over eightfold, to 86%. Mannitol, at 0.1 to 0.2 M concentration, stimulated the frequency of shoot regeneration in Pusa Basmati 1 by fivefold but had no effect in Basmati 385. Mannitol at 0.4 M concentration completely inhibited shoot regeneration but promoted embryogenesis. These calli regenerated shoots with greater frequencies when transferred to mannitol-free medium. Partial desiccation of rice calli resulted in an up to threefold increase in the shoot regeneration frequency. Best regeneration frequencies (54-98%) were obtained when 24 hdesiccated calli were grown on regeneration medium with 1.0% (w/v) agarose. A similar stimulatory effect of water stress on plant regeneration was observed in another Indica rice variety, IR43, and a Japonica rice variety, Taipei 309.

Agarose gel electrophoresis.

After digestion of DNA with a restriction enzyme (Chapter 50), it is usually necessary, for both preparative and analytical purposes, to separate and visualize the products. In most cases, where the products are between 200 and 20,000 bp long, this is achieved by agarose gel electrophoresis. Agarose is a linear polymer that is extracted from seaweed and sold as a white powder. The powder is melted in buffer and allowed to cool, whereby the agarose forms a gel by hydrogen bonding. The hardened matrix contains pores, the size of which depends on the concentration of agarose. The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.3 to 3%. Many different apparatus arrangements have been devised to run agarose gels; for example, they can be run horizontally or vertically, and the current can be conducted by wicks or the buffer solution. However, today, the "submarine" gel system is almost universally used. In this method, the agarose gel is formed on a supporting plate, and then the plate is submerged into a tank containing a suitable electrophoresis buffer. Wells are preformed in the agarose gel with the aid of a "comb" that is inserted into the cooling agarose before the agarose has gelled. Into these wells are loaded the sample to be analyzed, which has been mixed with a dense solution (a loading buffer) to ensure that the sample sinks into the wells.

Field and pore size dependence of the electrophoretic mobility of DNA: a combination of fluorescence recovery after photobleaching and electric birefringence measurements.

By combining an electrophoretic cell with a setup of fluorescence recovery after photobleaching (FRAP) we can measure the electrophoretic mobility mu of double-stranded lambda DNA in agarose gel as a function of electric field E and gel concentration C. Mobility varies linearly with the field in agreement with the biased reptation model with fluctuations. The slopes are analyzed in term of orientation and compared with birefringence results. The mobility extrapolated at zero field follows the prediction of the reptation theory; we deduced the variation of the pore size with the agarose concentration. With a special use of our setup, we measure directly the free-mobility mu 0 of the DNA.