Why does agarose form a gel

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.

Finally, the dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated. The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard against the distance traveled by each band. It is important to note that different forms of DNA move through the gel at different rates. Supercoiled plasmid DNA, because of its compact conformation, moves through the gel fastest, followed by a linear DNA fragment of the same size, with the open circular form traveling the slowest.

In conclusion, since the adoption of agarose gels in the s for the separation of DNA, it has proven to be one of the most useful and versatile techniques in biological sciences research. National Center for Biotechnology Information , U. J Vis Exp. Published online Apr Author information Copyright and License information Disclaimer. Correspondence to: Pei Yun Lee at ude. This article has been cited by other articles in PMC.

Abstract Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from bp to 25 kb 1. This allows them to keep the pH at a relatively constant level if the jar gets too low high pH protonated copies donate protons, and if it gets too full low pH , the deprotonated copies take some. And let it cool. As it cools, it goes from clear to cloudy as it transforms from a liquid to a gel.

These joined helices can still move around, but as a group. These rearrangements may require kicking out some water in the process. Chewing food compresses the meshes formed by gumming agents like agarose that give your food structure, expelling juiciness. And there are some things they can do to help prevent it.

To understand how these methods work, it might help to think about the gel a different way — kinda like a bunch of interconnected water balloons. If osmotic pressure wins, the water stays but if elastic pressure wins, the water leaves. But all this is no match for your jaws which add mechanical pressure that squeezes the balloons. If this happens, when you separate the products by size by running them through an AGAROSE GEL whose pores slow down bigger things you end up with bands of multiple sizes, only 1 of which is the band you want hopefully the biggest, brightest one!

But only in the 1st step of each cycle. So hunker down for a quick run down on the difference between polymerising and non-polymerising gel matrices. Of the common gel matrices used in molecular biology, polyacrylamide, agar and agarose, polyacrylamide is the one that polymerises. The polymerisation reaction, shown in the diagram below, is a vinyl addition catalysed by free radicals.

In the absence of bis-acrylamide, the acrylamide would polymerise into long strands, not a porous gel. But as the diagram shows, bis-acrylamide cross-links the acrylamide chains and this is what gives rise to the formation of the porous gel matrix. The amount of crosslinking, and therefore the pore size and consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bis-acrylamide.

For more information on polyacrylamide gel polymerisation see Biorad Bulletin So what about agarose? Well, agarose — the main component of the gelatinous agar that can be isolated from certain species of seaweed — is itself a polymer.