Turn on the power supply and verify that both gel box and power supply are working. Remove the lid. Slowly and carefully load the DNA sample s into the gel Fig. An appropriate DNA size marker should always be loaded along with experimental samples.
Replace the lid to the gel box. The cathode black leads should be closer the wells than the anode red leads. Double check that the electrodes are plugged into the correct slots in the power supply.
When electrophoresis has completed, turn off the power supply and remove the lid of the gel box. Remove gel from the gel box. Drain off excess buffer from the surface of the gel.
Place the gel tray on paper towels to absorb any extra running buffer. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done using a gel documentation system Fig. DNA bands should show up as orange fluorescent bands. Take a picture of the gel Fig. Figure 5 represents a typical result after agarose gel electrophoresis of PCR products.
After separation, the resulting DNA fragments are visible as clearly defined bands. The DNA standard or ladder should be separated to a degree that allows for the useful determination of the sizes of sample bands. In the example shown, DNA fragments of bp, bp and bp are separated on a 1. 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.
The locking tray system prevents the loss of samples from wells, and bands remain sharp because all the samples remain confined within the wells prior to electrophoresis down the gel.
Agarose gel electrophoresis is an excellent teaching tool for students in laboratory science classes from middle school through early college. A wide range of hands-on activities featuring agarose gel electrophoresis is amenable to typical class sizes and can be targeted to many different levels.
Topics can range from studying molecular sieving in order to determine which dyes are used in candies, to cloning, or the analysis of PCR products.
Bio-Rad has a wide range of products for agarose gel electrophoresis as well as an expanding number of kits that provide both educational and fun experiences.
A large library of support materials is available including manuals, curricula, and other resources to aid in teaching the theory and practice of molecular biology in a variety of applications.
Educational discounts are available for qualified educators. Be careful not to burn yourself, because it will be hot. Next add ethidium bromide -- this is a chemical that intercalates DNA and makes it visible under UV light.
You might want to wear gloves. The amount of EtBr to add is as follows: of a 0. The casting trays we have have this rubber stuff around the edges that makes a tight seal when you put them in the gel boxes sideways. They also have notches where you can insert a comb.
The size comb to use depends on the width you want your wells to be. It will take about 20 minutes for a small gel to harden enough to be used, longer for bigger gels.
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