Agarose gels

Electrophoresis running buffer Tris-borate-EDTA, Tris- 

Prepare the working solution of the running buffer (TBE or TAE buffer). Always use the same buffer for gel preparing and for electrophoresis. 

Add agarose (amount depending on the needed concentration). Heat with microwave or water bath until the agarose is dissolved in the buffer. 

Add suitable dye such as ethidium bromide (0.5 pg ethidium bromide/1 ml gel  

Pour the gel solution into a suitable electrophoresis tray, to give a 3-5 mm gel, insert the comb, leave the gel for 30 min. 

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Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Both the ease of use of this method for characterization of proteins and nucleic acids, and the abiHty to analyze many samples simultaneously for comparative purposes, have led to the prevalence of this technique. The drawbacks of a polyacrylamide matrix is that acrylamide is a neurotoxin, the reagents must be combined extremely carefiiUy, and the gels are not as pHable as most agarose gels. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

The double-immunodiffusion technique, often referred to as the Ouchtedony technique, uses an agarose gel as the matrix. Holes are made in the agarose where either sample or antisera is placed. The two solutions are allowed to diffuse into the matrix for a predetermined time. If there is a reaction... 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Plus One REPEL-SILANE ES (a solution of 2% w/v of dichloromethyl silane in octamethyl cyclooctasilane) is used to inhibit the sticking of polyacrylamide gels, agarose gels and nucleic acids to glass surfaces and is available commercially (Amersham Biosciences). 

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

Like other GFC matrices, including TSK-GEL SW and TSK-GEL PW packings, and dextran and agarose gels, Toyopearl HW resins exhibit some ionic and hydrophobic interaction with samples. The hydrophobic properties of Toyopearl HW resins, however, can be utilized more effectively for improved protein purifications because, unlike numerous other GFC packing materials, Toyopearl HW resins can be used with high levels of organic solvent (38). 

Purifications are made simpler with Toyopearl HW media because there is no risk of leached polysaccharides to contaminating eluted fractions. Saccharide derivatives have been known to leach from conventional low-pressure column packings, such as dextran or agarose gels. 

Agarose gels have been used for more than two decades to separate polysaccharides (17-22). In particular, Sepharose CL 2B is widely used (6-8) to separate native starch, but continuously improved mechanical and chemical stability made all of the Sepharose CL gels perfect systems for the analysis of high molecular and broad distributed polysaccharides (23-28). 

Prepacked columns with cross-linked high-resolution (HR) agarose gels provide a high number of theoretical plates and fast separations (29,30).The Superose gel material of Pharmacia Biotech is a highly epichloro-hydrine cross-linked... 

Recently, the separation of some milligram quantities of terbutaline by classical gel electrophoresis has been reported [194]. A sulfated cyclodextrin impregnated on the agarose gel was used as a chiral selector and the complete resolution was achieved in 5 h. Analogously, small amounts of enantiomers can be isolated using thin-layer... 

Table 11-3. Migration times for piperoxan enantiomers for four eonseeutive runs on the same agarose gel using sulfated eyelodextrin as the ehiral seleetor.

Small-angle X-ray scattering (SAXS), circular dichroism (CD), and UV spectroscopy at different temperatures were used to investigate the nature of calf-thymus DNA in aqueous solution, in the presence of [Me Sn] " (n = 1-3) species. The results demonstrate that the [MeSn(IV)] moiety does not influence the structure and conformation of the DNA double helix, and does not degrade DNA, as indicated by agarose gel electrophoresis. Inter alia, the radii of gyration, Rg, of the cross section of native calf-thymus DNA, determined by SAXS in aqueous solution in the presence of [Me Sn] " (n = 1-3) species are constant and independent of the nature and concentration of the [Me Sn] species. 

E.coli K12 TGI were grown to log phase (up to OD6oo=0.20-0.30) in Luria-Bertani (LB) broth, washed and ultimately concentrated 25 times in ice-cold 100 mM of CaCb. DNA was extracted from agarose gel after electrophoresis, added to 200 ml of competent cell and incubated at 0°C for 15 min. The cell-DNA complex was transferred to 42°C for exactly 90 s and was rapidly chilled in ice. Then 1000 ml LB-broth was added and the cells were incubated at 37°C for 60 min. 100 ml cells was spread on LB-agar with and without selective marker ampicillin (50 mg/ml), to obtain the number of transformants and viable cells respectively. Plates were incubated at 37°C for 18-24 h. 

Fig. 3. Agarose gel after electrophoretic separation of amplified DNA of Chlamydia trachomatis without (A), and with the addition of Ce-AR (B) in the electrophoretic system at exposure by transilluminator at 254 nm for 5 (1), 30 (2), 300 (3) and 600 (4) seconds, and profiles of the electrophoretic mobility (C).

The used variants of Cg-AR application were adding to the wells of the gel to DNA, directly bringing into the agarose gel and the addition to electrophoretic buffer. The use of the latest way demonstrated its greatest efficiency by saving up to 1.63 times more DNA preparations if compared with the standard method of electrophoresis, while other ways showed 15.45% increase when Cg-AR was introduced into an agarose gel and 1.63%- when added to the DNA preparations. 

Grendemann, D., Schumig, E. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques, Vol.21, No.5, (November 1996), pp. 898-903, ISSN 0736-6205... 

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

FIG. 4 Chemical composition and physical structure of agarose gels. (Reprinted by permission of Wiley-VCH and P. Serwerfrom Ref. 350, Copyright 1983, WUey-VCH.)... 

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