Why do we use agarose gel in electrophoresis

The speed of movement through the gel is then determined by the voltage gradient, i. The exact value depends on your samples and should be determined empirically. To apply this electrical field, we use a DC power supply. Most electrophoresis power supplies can be set to provide either a constant current or a constant voltage, with each having advantages and disadvantages. However, since agarose gel electrophoresis uses a continuous buffer system i.

We can simply set the power supply to constant voltage, based on the size of the tank as described above. One potential issue is the production of heat due to the flow of current through the system which can be especially high with larger tanks that require higher voltage. For this reason, it is advisable to use some form of cooling, either passive in the form of a cooling block , or active such as a recirculating chiller , for larger electrophoresis systems.

After the DNA has migrated through the gel, it needs to be visualised, so we can determine the length and abundance of the molecules in the sample. Since DNA is not visible to the naked eye, we stain it with either a coloured stain, such as methylene blue, or more frequently with a fluorescent stain such as Ethidium Bromide.

Fluorescent stains give much better detection levels when imaging gels. There are a wide range of stains on the market, some being added to gel before casting and some being used to stain the gel after the run. Whichever stain you use, the next step is to capture an image in a gel documentation system. Gel documentations systems , or gel docs for short, use high sensitivity cameras to capture images of the agarose gels. Often these systems are equipped with UV or blue light transilluminators, which are used to excite the fluorescent stains, which then emit light which can be captured by the camera.

All gel docs will come with some form of illumination source, a filter to remove background light and a camera to detect the signal. Other than these basics there are a huge range of gel docs available starting from basic hood systems to systems with integrated PCs and touchscreens. Given the simple nature of this technique, scientists have been able to apply it to a wide range of studies, some of which are discussed below.

Probably the most frequent application of agarose gel electrophoresis is in molecular cloning. This is the construction of recombinant DNA molecules that are integrated into various organisms to create genetic modifications. The purpose of these modifications varies and can include production of a specific biomolecule, for example the production of insulin in pharmaceutical manufacturing.

Other applications of molecular cloning include adding fluorescent protein fusions to existing cellular proteins to study their location in cells and creating new genetic circuits to carry out specific functions, such as breaking down toxins. Whatever the desired end product is, electrophoresis is a key step in both the production and quality control of DNA fragments used in molecular cloning.

Electrophoresis can be used to analyse the fragments created by polymerase chain reaction PCR or restriction digest, to ensure they are of the correct size. It can also be used to purify fragments, by running them on the gel and subsequently cutting out the band of interest and purifying the DNA from the agarose.

Combined with PCR, agarose gel electrophoresis can be a powerful technique for identifying individuals based on their genetic code. The human genome contains many regions of short repeats, the number of which vary uniquely between individuals. By targeting these regions with specific PCR primers, a profile of band on an electrophoresis gel corresponding to these regions can be created that is unique to that individual. 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?

What is DNA sequencing? How helpful was this page? What's the main reason for your rating? This technique has lots of applications. Generally speaking you can analyze DNA fragments that result from an enzyme digestion of a larger piece of DNA to visualize the fragments and determine the sizes of the fragments. In addition to its usefulness in research techniques, agarose gel electrophoresis is a common forensic technique and is used in DNA fingerprinting.

Ethidium bromide is an intercalating dye, which means it inserts itself between the bases that are stacked in the center of the DNA helix. One ethidium bromide molecule binds to one base. As each dye molecule binds to the bases the helix is unwound to accommodate the strain from the dye. Ethidium bromide can easily get into your cells. Human DNA is linear and stains well. This means that it can get into your DNA and untwist it. This is not a good thing, so make sure you are careful and protected when using ethidium bromide.

Another advantage of using Bio-Rad precast gels for agarose gel electrophoresis is that the running tray locks into place in Mini-Sub cell GT gel boxes. Using this system, agarose gels cannot shift during sample loading, putting the lid on the box, or the initial stages of electrophoresis. 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.