Why gel electrophoresis

The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample. Preparing the DNA for electrophoresis A dye is added to the sample of DNA prior to electrophoresis to increase the viscosity of the sample which will prevent it from floating out of the wells and so that the migration of the sample through the gel can be seen.

The fragments in the marker are of a known length so can be used to help approximate the size of the fragments in the samples. The prepared DNA samples are then pipetted into the remaining wells of the gel. When this is done the lid is placed on the electrophoresis tank making sure that the orientation of the gel and positive and negative electrodes is correct we want the DNA to migrate across the gel to the positive end.

Separating the fragments The electrical current is then turned on so that the negatively charged DNA moves through the gel towards the positive side of the gel. Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run.

The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye. The electrical current is left on long enough to ensure that the DNA fragments move far enough across the gel to separate them, but not so long that they run off the end of the gel. Related Content:. What is DNA? What is PCR polymerase chain reaction? See Subscription Options. Discover World-Changing Science.

He replies: "DNA is a charged molecule. Get smart. Sign Up. Support science journalism. Knowledge awaits. 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.

In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. As previously mentioned, gel electrophoresis involves an electrical field; in particular, this field is applied such that one end of the gel has a positive charge and the other end has a negative charge.

Because DNA and RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel.