Figure 5. An image of a gel post electrophoresis. EtBr was added to the gel before electrophoresis to a final concentration of 0. The gel was exposed to uv light and the picture taken with a gel documentation system. Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose's high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of agarose 7 in the gel matrix.
In general, the higher the concentration of agarose, the smaller the pore size. Traditional agarose gels are most effective at the separation of DNA fragments between bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis 6 , which involves the application of alternating current from two different directions. In this way larger sized DNA fragments are separated by the speed at which they reorient themselves with the changes in current direction.
DNA fragments smaller than bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and have better resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application of high voltages, thereby enabling the separation of DNA fragments and the determination of DNA sequence quickly.
Agarose can be modified to create low melting agarose through hydroxyethylation. Low melting agarose is generally used when the isolation of separated DNA fragments is desired.
Hydroxyethylation reduces the packing density of the agarose bundles, effectively reducing their pore size 8. This means that a DNA fragment of the same size will take longer to move through a low melting agarose gel as opposed to a standard agarose gel.
Because the bundles associate with one another through non-covalent interactions 9 , it is possible to re-melt an agarose gel after it has set.
EtBr is the most common reagent used to stain DNA in agarose gels When exposed to uv light, electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy light as the electrons return to ground state. EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner.
EtBr is a suspect mutagen and carcinogen, therefore one must exercise care when handling agarose gels containing it. In addition, EtBr is considered a hazardous waste and must be disposed of appropriately. Of these, Methyl Blue and Crystal Violet do not require exposure of the gel to uv light for visualization of DNA bands, thereby reducing the probability of mutation if recovery of the DNA fragment from the gel is desired.
However, their sensitivities are lower than that of EtBr. Moreover, all of the alternative dyes either cannot be or do not work well when added directly to the gel, therefore the gel will have to be post stained after electrophoresis.
Because of cost, ease of use, and sensitivity, EtBr still remains the dye of choice for many researchers. However, in certain situations, such as when hazardous waste disposal is difficult or when young students are performing an experiment, a less toxic dye may be preferred.
Loading dyes used in gel electrophoresis serve three major purposes. First they add density to the sample, allowing it to sink into the gel. Second, the dyes provide color and simplify the loading process. Furthermore, several modifications of agarose gel electrophoresis by adding a special reagent in the agarose gel are proposed [ 7 , 8 ].
The principle of these modified electrophoresis methods is basically the same as the traditional method described here, and the cost-saving method in this manuscript will also be applicable for such modified methods. Agarose gel electrophoresis is a very popular experiment for training of students in educational institutions [ 9 , 10 , 11 ].
Cost-effective methods described here should be good news for such institutions, because running costs cannot be ignored in student training practices. Agarose is a kind of carbohydrate macromolecules polysaccharides , also known as a kind of dietary fibers. Agarose is purified from a certain red seaweed Rhodophyta.
Polysaccharides from Rhodophyta mainly consist of agarose and agaropectin. The quality of agarose depends on such purification steps, and these steps push up agarose commercially much expensive. Agar is also a well-known gelling reagent for bacteria medium.
It can be said that agar for bacteria medium is a partially and roughly purified form of sea weeds and is of lower quality than agarose for electrophoresis. In my experience, agar for bacteria medium is quite suitable for a gel electrophoresis reagent. Nagano, Japan —Funakoshi Co. Tokyo, Japan Figure 1 , left is an agar of which grade is for bacteria, but its quality is very good for electrophoresis. Nagano, Japan Figure 1 , right is an agar for cooking, the quality of which is adequate for agarose gel electrophoresis.
Although there is no warranty or trust for results i. Agars for not electrophoresis but other use. Right, INA S-7 agar for cooking. INA agar BA for agarose gel electrophoresis. Result of the electrophoresis by using INA S-7 agar. Generally, agarose of low purity is more breakable because of its low gel strength. This disadvantage was critical especially when Southern or northern blotting was achieved in the experiment. In recent days, such blotting techniques have given way to the other; for example, polymerase chain reaction PCR to see DNA polymorphism and real-time PCR to see gene expressions.
The major visualizing way of DNA in agarose gel is to use ethidium bromide EtBr or the other DNA intercalators that make fluorescence excited in certain wavelength [ 12 ].
Several protocols for staining reagent to intercalate DNA are known; a add the reagent in the gel before solidifying, b add the reagent in the loading buffer at electrophoresis, and c soak the gel in the reagent buffer after electrophoresis Figure 4. In a and b , a photograph of the gel can be taken with a gel tray, when the tray is clear.
In such a situation, a low gel strength does not disturb electrophoresis and DNA visualization. Based on these reasons, a low gel strength seems not a too much annoying point when simply doing electrophoresis and taking photographs.
Scheme for staining and visualizing of DNA in agarose gel electrophoresis. It is known that used agarose gel is reusable again and again. Recycling of agarose after electrophoresis is very effective for cost-saving. Several reports are published [ 13 , 14 ], in which used agarose gels are simply boiled and poured to a gel tray. After cooling to make the recycle gel solid, the recycled gel is enough for applying another electrophoresis.
EtBr is well known as toxic mutagen [ 15 , 16 ], so when used and stained gel is boiled, toxic fumes containing EtBr appear. This fume will be hazardous when incorporated through the respiratory system. To avoid such hazardous fumes, a freeze-and-thaw of used agarose gel is very effective for removing toxic EtBr from the gel [ 17 ].
By repeating freeze-and-thaw, the EtBr concentration of used agarose gel dramatically reduces to as much as a negligible level. Additional images from Wikimedia via Jacopo Werther electrophoresis machine and Mnolf gel electrophoresis. Guruatma "Ji" Khalsa. Agarose Gel Electrophoresis. Scientists, teachers, writers, illustrators, and translators are all important to the program.
If you are interested in helping with the website we have a Volunteers page to get the process started. Digging Deeper. Digging Deeper: Depression and the Past. Digging Deeper: Germs and Disease. Digging Deeper: Milk and Immunity. Couldn't you just dye? Although polyacrylamide gel electrophoresis PAGE can deliver a higher resolution than agarose gel electrophoresis that is, PAGE can provide a cleaner separation of molecules of different sizes , agarose gel electrophoresis has several important advantages: a single gel can separate a much broader range of molecular sizes, nucleic acids are easy to visualize with fluorescent staining, and the extraction of DNA bands for subsequent purification and use is simpler and more efficient.
There are many benefits to using precast agarose gels. The principal advantages of precast gels include both speed and the assurance of consistent run conditions across loadings and between experiments. This consistency is particularly useful when screening for small changes or comparing a large number of samples. Bio-Rad precast agarose gels provide high-resolution separation of DNA fragments from 20—20, bp long.
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