Electrophoresis is the movement of charged particles through an electrical field. Since the sugar-phosphate backbone of DNA* has a negative charge, electrophoresis can be used to pull DNA through an electrical field towards the positive electrode of a circuit. Molecular biologists have exploited this behavior to develop techniques that separate, clean and analyze DNA fragments. There are an enormous number of variations of gel electrophoresis including SDS-PAGE, DNA sequencing, 2D-gel electrophoresis, DGGE and many many others. The details of each of these technique differ but they all exploit the fact that charged particles such as DNA migrate when placed in an electrical field. And, that the direction of migration depends on the charge on the particle.
This illustration shows the different components of the gel electrophoresis set-up and describes the steps required to prepare a gel and DNA samples for analysis using this technique.
Click on the objects and steps to navigate through the process of setting up a gel electrophoresis :
At the heart of the technique is the gel. It is a matrix that contains pores though which the DNA is drawn when an electrical current is applied. Without a gel, all of the DNA would go right to positive electrode (called the anode). The size of the pores control the rate at which the DNA moves. The smaller the pores, the slower the DNA moves. The length of the DNA fragments influences the rate at which they are pulled through the gel. Longer fragments moving more slowly.
A number of different matrixes are used for electrophoresis. Agarose is one of the most common. Agarose gels are non-toxic, relatively inexpensive and easy to prepare. The higher the concentration* of agarose in the gel, the smaller the pores. A relatively high concentration of 1% agarose is used to separate small DNA fragments while lower concentrations are used for separating large fragments. For more exacting work, or for the separation of larger DNA fragments, polyacrylamide can be used. Polyacrylamide provides higher resolution relative to agarose and can be use in a larger variety of conditions but has the drawback of being toxic.
Wells are small indentations created in the gel when it is made. The wells are uniformly spaced along the side of the gel closest to the negative electrode. The even, linear spacing of the wells provides a uniform starting position for the samples. The wells also allow the samples to be placed into the gel so that when current is applied, the samples are pulled through the middle of the gel, not across the top.
A solution is used to carry the electrical current though the gel and help maintain a constant environment during the run. The solution is called a running buffer. The buffering is needed to maintain a constant pH and provide ions in the solution to facilitate the flow of electricity. Heat is generated by the application of a current to the gel, the running buffer also helps keep the gel cool. This is especially importation for agarose gels because they melt if they get too hot.
The Gel Box
The gel box is the container that holds the the gel submerged in running buffer. It is designed so that when current is applied through the electrodes attached to the box, the current flows through the gel creating the electrical field needed to push the negatively charged DNA molecules towards the positive electrode.
Power and Power Supply
The force needed to draw the DNA though the gel is provided by electricity. A power supply takes the the standard alternating-current electricity available from a wall outlet and converts it into the one way, direct-current needed to set up an electrical field across the gel. Power supplies also provide a mechanism to control the amount and force (amperage and voltage) contained in the field. The lower the voltage, the slower the DNA will migrate.
Given enough time, all of the DNA in a sample will eventually run to the end of the gel and out into the surrounding buffer. This makes the amount of time the current is on is an important parameter. Most power supplies have a timer to turn the power after a predetermined interval.
Sample and Sample Preparation
A variety of different materials are analyzed with gel-based electrophoresis techniques. For the purposes of this discussion we are assuming that the samples are linear strands of double stranded DNA. In this case the primary factor influencing the migration of the DNA strands is their length. Other types of material that are commonly run on gels are DNA plasmids, RNA and proteins.
The Loading Dye*
Loading dye is a colored buffer mixed with the DNA prior to loading onto the gel. The loading dye contains a relatively high concentration of either glycerol or sucrose. This makes the solution more dense than the surrounding running buffer so that when a sample is pipetted over top of a well it sinks down into the well. It also contains a small amount of dye (typically bromophenol blue). Coloring the sample provides quick conformation that the samples have sunk into the wells and makes it easy to keep track of which wells have already been loaded.
At the pH range in which the gels are buffered, bromophenol blue has a negative charge so it migrates in the same direction as the DNA. This has the additional benefit of providing visual indication of the progress of the DNA migration. This is extremely useful because the DNA itself is not visible during the running of the gel. Visualizing the DNA after the gel has been run requires a separate step that involves staining the gel with something that binds with the DNA, rendering it visible.
Due to the many factors that affect the rate of migration of DNA through the gel, estimating the exact length of a band in the gel must be done relative to the position of other bands in the same gel. A standard (also often referred to as a DNA ladder) is placed in one of the wells. By comparing the movement of the fragments of known length in the standard with the fragments in the samples, an accurate estimate of the length of the DNA strands in the samples can be made. The standard is treated the same way as the samples: mixed with loading dye and added to one of the wells in the gel.
With all of the materials above in hand, the steps required for setting up a gel are:
- Prepare the gel box by adding enough running buffer so that the gel will be completely submerged once it is place in the gel box. The buffer does not have to be replaced every time a new gel is run, but the electrophoresis process does degrade the buffer so it is a good practice to replace it frequently.
- Place the gel in the gel box making sure the gel is completely submerged in the buffer and that the wells are oriented properly (closest to the negative, usually black, electrode).
- Add loading dye to the samples and standards.
- Pipette a small volume* of sample/standard into each well.
- Connect the power supply electrodes to either end of the gel box.
The gel is ready to run.
Many factors influence the migration of charged particles in an electrical field. In order for DNA gel electrophoresis to work as a way to consistently separate DNA polymers based on their length, conditions are manipulated in order to create as constant an environment as possible. The factors that influence migration include the ionic composition and pH of the running buffer; the temperature of the gel; the voltage applied to the gel and the porosity of the gel matrix. By controlling for all of these other factors, gel electrophoresis can be used to separate DNA strands based on their length.
While the gel type, pre and post processing and factors that influence migration direction and rate vary from application to application, a solid understanding of the basic agarose gel electrophoresis of linear strands of DNA described above provides the foundation upon which an understanding of the other electrophoresis techniques can be built.
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