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Ncbe Briefing Gel Electrophoresis No. 1 | JANUARY 2017 NCBE BRIEFING GEL ELECTROPHORESIS Gel electrophoresis is a key technique in modern biology that features in all of the new A-Level Biology specifications in England. It is a way of separating DNA, RNA or proteins based on their size and/or the electrical charge on the molecules. DNA gel electrophoresis Visualising DNA For DNA gel electrophoresis, a gel is cast from After electrophoresis, the DNA is visualised. In agarose, dissolved in buffer solution. Agarose is a very research laboratories, a fluorescent dye will have pure (and expensive) form of agar, which is obtained been incorporated into the agarose gel before it from seaweed. At one end of the slab of gel are several was cast. After the gel has been ‘run’ it is illuminated small wells, made by the teeth of a comb that was with ultraviolet (UV) light and the dye, which binds to placed in the molten agarose before it set. A buffer DNA, shows up as bright fluorescent bands. Ethidium solution is poured over the gel, so that it fills the wells bromide was until recently the most commonly used and makes contact with the electrodes at each end of DNA stain. Ethidium bromide has similar dimensions the gel. Ions in the buffer solution conduct electricity. to a base pair in DNA. When ethidium bromide binds to DNA, it slips between adjacent base pairs and The test samples (DNA fragments) are mixed with a stretches the double helix. This causes errors when small volume of loading dye. This dye is dissolved in a the DNA is replicated. dense sugar solution, so that when it is added to the wells, it sinks to the bottom, taking the DNA sample with it. An electrical potential is applied across the gel. Phosphate groups give the DNA fragments a negative electrical charge, so that the DNA migrates through the gel towards the positive electrode. Small DNA fragments move quickly through the porous gel; larger molecules travel more slowly. In this way the pieces of DNA are separated by size. The loading dye also moves through the gel, so that the progress of the electrophoresis can be seen (the DNA itself Above: Intercalation of ethidium bromide between two adjacent bases in a DNA molecule. is invisible). Short-wavelength UV light is itself harmful and ethidium bromide’s breakdown products are thought to be potent mutagens and carcinogens. Ethidium bromide should therefore not be used in schools*. For reasons of safety and because UV light of this wavelength causes unwanted mutations in the DNA being studied, several alternative stains are now often used in research labs. These include SYBRsafe® and GelRed®, which although they are thought to be safer than ethidium bromide, are far more expensive [Ethidium bromide costs £4.50 per mL compared with £133 per mL for SYBRsafe® and £200 per mL for www.ncbe.reading.ac.uk 1 Copyright © NCBE, University of Reading, 2017 NCBE BRIEFING GelRed® (2016 prices).] An additional advantage of N some of these compounds is that they will fluoresce Azure A + (H C) N NH.CH in blue, rather than harmful UV light. 3 2 S 3 Cl- In schools, safer, cheaper dye solutions are used N Azure B to stain the entire gel, including the DNA, after + (H3C)2N S NH2 electrophoresis. Suitable stains include Azure A and Cl- Azure B, Toluidine blue O and Nile blue sulphate. This type of stain is not thought to intercalate within the N CH3 DNA double helix, but instead binds ionically to the + Toluidine blue O (H3C)2N S NH2 negatively-charged phosphate groups of the DNA. Cl- O NH2 (CH2CH3)2N Such dyes are not as sensitive as ethidium bromide + Nile blue sulphate 1 SO -2 and the newer fluorescent dyes, and some of them 2 4 N may colour the gel heavily. Consequently, prolonged ‘destaining’ in water may be necessary before the Above: Some dyes that are thought to bind ionically DNA bands can be seen. Methylene blue, which is to DNA. sometimes used for staining DNA on agarose gels in schools, is far from ideal, as it requires destaining and it fades rapidly after use. HOW MUTAGENIC IS Although these alternatives to ethidium bromide ETHIDIUM BROMIDE? are thought to be relatively safe, they have not been intensively studied for long-term effects and the In recent years there has been some mechanisms by which they bind to DNA are not fully controversy about the dangers of ethidium understood. As with all laboratory chemicals, suitable bromide. The compound is used as a drug safety precautions should be exercised when handling for treating cattle with trypanosomiasis any dyes, particularly when they are in dry, powdered (African sleeping sickness) at far greater form. concentrations than are used in the lab. The cattle do not seem to suffer any adverse effects, but since the animals are usually Viewing gels slaughtered after a few years, any long-term harm would not be noticed. When ethidium Gels stained with a blue dye such as Toluidine blue O bromide is metabolised in the liver, the can be viewed in daylight. A smartphone with a white compounds produced are highly mutagenic. background light (some ‘torch’ apps are suitable) can It is probably correct to say that ethidium be used as a ‘lightbox‘. Alternatively, LED lightboxes bromide is not as harmful as some people sold for tracing can be used. A yellow-coloured filter think it is, but it should still be handled with may help to enhance the contrast when photographing care and disposed of correctly. The relevant gels that have been stained with blue dyes. safety regulations state that it MUST NOT be used in UK schools*. Storing stained gels Gels stained with Toluidine blue O or Azure A can be H2N NH2 stored refrigerated in a plastic bag to prevent them + from drying out. Provided they are not exposed to N Br - light, gels kept like this will not fade for many months. Ethidium C2H5 bromide * See: www.ncbe.reading.ac.uk/SAFETY/dnasafety. html www.ncbe.reading.ac.uk 2 Copyright © NCBE, University of Reading, 2017 No. 1 | JANUARY 2017 EFFECT OF VOLTAGE Fragment What’s the best voltage to use? size (kb) At low voltages, movement of linear DNA is proportional to the voltage 23.13 applied. As the voltage is increased, the mobility of the higher molecular mass fragments is increased differentially (the larger fragments tend to 9.42 ‘catch up’ with the smaller ones). Hence the effective range of separation 6.56 is decreased as the voltage is increased. For the best resolution, 0.8% 4.36 agarose gels should be run at no more than 5 V per cm (as determined by the distance between the electrodes). The NCBE electrophoresis 2.32 equipment, which is designed to work at 36 V, has a distance between the 2.03 electrodes of ~85 mm, which is close to the optimum. 0.56 Calculating the resolution of a gel For λ DNA digested by HindIII (shown on the left), the resolution can be 5 V / cm 20 V / cm calculated by dividing the distance between the 23 and 2 kb fragments by Good Poor the total distance travelled by the 2 kb fragment. separation separation EFFECT OF GEL CONCENTRATION Gel concentration also affects the movement of DNA fragments. There is a linear relationship between the logarithm of the mobility of the DNA and the gel concentration. By altering the agarose concentration it is possible to control the range of sizes of fragments that can be separated by electrophoresis. The example on the left shows λ DNA digested by HindIII. The optimum gel concentration for separating these λ DNA fragments is ~0.8% (w/v), which is the concentration suggested in the NCBE’s Lambda protocol module (see page 6). For larger or smaller DNA fragments, a different agarose concentration may be better. For instance, to show chloroplast DNA fragments of between ~500 and several thousand base-pairs, such as those produced by the NCBE’s PCR and plant evolution module, an agarose concentration of 1.5% (w/v) is recommended. The table below shows the concentration of agarose needed for separating DNA fragments of different sizes. 0.5 1.0 1.5 2.0 Gel concentration (%) Agarose Separation range Relative Above: Even a small difference (% w/v) (kb) gel strength in gel concentration can have a 0.3 60–5 Very weak significant effect on the quality of the results you see. For that 0.6 200–1 Weak reason, it is important to make 0.7 10–0.8 Moderate up agarose solutions accurately, 0.9 7–0.5 Moderate using buffer, not water. Don’t try to weigh out small amounts of 1.2 6–0.4 Strong agarose, make up a large volume: 1.5 4–0.2 Strong it will keep indefinitely in a sealed container. 2.0 3–0.1 Strong Copyright © NCBE, University of Reading, 2017 3 www.ncbe.reading.ac.uk NCBE BRIEFING Polyacrylamide gels and protein It is also possible to separate proteins using a special electrophoresis type of agarose, but in contrast to the procedure using polyacryamide, with agarose the proteins are To separate proteins by electrophoresis, gels cast separated by electrical charge only (not charge and from polyacrylamide are sometimes used. Before size). This is because the pores within the agarose proteins are run on a gel, they are treated with a strong gel are relatively large and the proteins can easily pass detergent, sodium dodecyl sulphate (SDS). This, through them. coupled with heating, causes the tightly-folded protein molecules to unfold and become linear, so that they As with DNA, the proteins on the gel are stained will move through the gel according to their sizes, not with an appropriate dye.
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