Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels

The electrophoresis of a series of DNA fragments ranging in size from 0.5 to 12 kilobase pairs, has been studied as a function of agarose gel concentration and electric field strength. The apparent mobility of all fragments decreased with decreasing electric field strength and with increasing gel concentration. When extrapolated to zero electric field strength and zero agarose concentration, the apparent mobility of all DNA fragments extrapolated to a common value (2.0 ± 0.1) × 10^?4 cm²/V s. The square roots of the retardation coefficients of the various fragments were found to be linearly related to the root-mean-square radii of gyration of the fragments, as predicted by pore-size distribution theory. As predicted by reptation theory, the molecular weights of the various fragments were found to be linearly related to the reciprocal of the apparent mobilities. An equation is given for estimating the apparent pore size of agarose gels between 0.25 and 1.5% in concentration.     

A simple and effective SuperBuffer for DNA agarose electrophoresis

In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25 V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

Improved resolution of DNA fragments in polysaccharide-supplemented agarose gels

The electrophoretic separation of nucleic acids, including small DNA fragments in the range 50-1000 bp, is presently carried out in polyacrylamide gels or in gels containing high concentrations of agarose. We have developed an alternative gel matrix composition which is inexpensive, nontoxic, easy to prepare, and highly transparent to visible and uv light. The composition combines a soluble nonionic polysaccharide such as hydroxyethylcellulose, methylcellulose, or galactomannan with a minimum but sufficient concentration of agarose to form a gel which immobilizes the "liquid phase sieve." These mixtures do not replace polyacrylamide for resolving fragments smaller than approximately 75 nucleotides. However, the new gels show DNA fragment resolution (band separation versus distance traveled) and optical clarity superior to those of conventional agarose.     

The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA concentrations of 200 micrograms/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800 micrograms/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 micrograms/ml (10(7) diploid human cells/ml agarose insert).     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

Quantitative isolation of DNA restriction fragments from low-melting agarose by Elutip-d affinity chromatography

The use of a disposable affinity column and low-melting-temperature agarose for the quantitative preparation of DNA restriction fragments is presented. After electrophoretic separation of DNA, the band(s) are excised and the DNA/agarose melted in a low-salt buffer. After cooling, the DNA is bound to an Elutip-d affinity column. Fragments are eluted at high salt and concentrated by ethanol precipitation. Recoveries greater than 80% are achieved with purity suitable for most applications in molecular biology.     

Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.     

DNA gel electrophoresis: effect of field intensity and agarose concentration on band inversion

We study the effect of electric field intensity and agarose gel concentration on the anomalous electrophoretic mobility recently predicted by the biased reptation model and experimentally observed for linear DNA fragments electrophoresed in continuous electric fields. We show that high fields and low agarose concentrations eliminate the physical mechanism responsible for anomalous DNA mobility and band inversion, in good agreement with theory, thus restoring the monotonic mobility-size relationship necessary for unambiguous interpretation of the results of DNA gel electrophoresis.     

Unexpected loss of genomic DNA from agarose gel plugs

Intact chromosomal DNAs are routinely prepared by embedding cells in agarose plugs before lysis. The large sizes of the genomic DNAs cause their retention while other macromolecules diffuse into and out of the gel matrix during lysis, washing and restriction cleavage incubations. However, in an analysis of agarose-embedded chromosomal DNAs cleaved with restriction enzymes, fragments larger than 30 kilobases were found to have eluted from the gel plugs. Since loss of fragments from gel plugs may affect qualitative and quantitative interpretations of electrophoretic patterns, an analysis of the diffusion of DNA segments from agarose plugs was performed. The two variables monitored were the time dependence and the DNA fragment size dependence of the diffusion process. The results indicate that small fragments (less than or equal to 2 kilobases) are quickly lost from 1% agarose gel plugs; moreover, significant amounts of large DNA segments (i.e., the 48.5-kilobase lambda phage chromosome) are also lost. In addition to urging caution in the analysis of restriction cleavage data, these observations suggest that intact small organelle genomes and extrachromosomal DNAs also may be lost from genomic DNAs prepared in agarose gel plugs.     

A simple, efficient, and economical method for recovering DNA from agarose gel

A simple method of recovering DNA from agarose gel that is fast, inexpensive, and friendly both to operators and environment is described. Two rows of wells are made in an agarose gel, and a DNA sample is loaded into the well nearest to the negative pole for separation by electrophoresis. Recovery is accomplished by pipetting the DNA-containing TAE buffer from the well near the positive pole after target DNA fragments have migrated into the well. A recovery rate of up to 94 +/- 2.3% was observed with this method.     

Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction

A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.     

Measurement of DNA length by gel electrophoresis. I. Improved accuracy of mobility measurements using a digital microdensitometer and computer processing

The electrophoretic mobilities of DNA polymer fragments in an agarose gel have been measured from a photograph of the gel by different methods and converted to lengths by the reciprocal method. The method of measurement can introduce large errors in the length estimates. The use of a digital microdensitometer to obtain optical density profiles of gel tracks with subsequent computer processing to find peak positions was found to give the most accurate DNA lengths.     

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 g DNA ml bacterial culture.     

Properties of agar: parameters affecting gel-formation and the agarose-iodine reaction

The effects of concentration of agarose, methyl sulphoxide, and substituted agaroses on the mechanism of gel formation have been evaluated with reference to the “Network theory of gel formation”. Factors affecting formation of the agarose gel-iodine color complex were investigated, and the results suggest that the iodine molecules are incorporated between the agarose helices in the Gel II state of agarose.     

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.     

Cleavage of DNA from 2 phages of Bacillus thuringiensis by EcoRI and HindIII endonucleases

The DNA of two Bacillus thuringiensis phages was restricted by endonucleases EcoRI and HindIII and the electrophoretic distribution of the fragments in agarose gel was studied. EcoRI was shown to restrict the DNA of phage 1-97A into 8 fragments and the DNA of phage 1-97B into 12 fragments. Restriction with HindIII results in the formation of 22 and 9 fragments for phage 1-97A and phage 1-97B, respectively. The molecular mass of the DNAs determined by summing up EcoRI restricts is 80.87 MDa for phage 1-97A and 32.45 MDa for phage 1-97B.