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Agarose gel electrophoresis
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate further than longer ones.
Additional recommended knowledge
The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered.
The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.
Factors affecting migration
The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of the DNA molecule is also a factor. To avoid this problem linear molecules are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs.
Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm).
Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid.
Visualisation: EtBr and dyes
The most common dye used for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible.. EtBr is a known carcinogen, however, and alternatives are available.
SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25 times more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or toxicity in humans.
SYBR Safe is a varient of SYBR Green that has been shown to have low enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more expensive.
Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Xylene cyanol and Bromophenol blue are typically used. They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp.
Gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases). However, it is normally used in a range of 100 bp to 20 kbp. Typical run times are about an hour.
Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules are only able to move end-on in a process called "reptation" and are more difficult to separate. In general higher concentrations of agarose are better for larger molecules; it will exaggerate the distances between bands. The disadvantage of higher concentrations is the long run times (sometimes days). Instead these gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.
Agarose is purified from agar, a gelatinous substance isolated from seaweed or human waste. Different purities of agarose are commercially available as are agaroses with different melting properties. High purity low melt agarose is often used if the DNA is to be extracted from the gel.
There are a number of buffers used for agarose electrophoresis. The most common being: tris acetate EDTA (TAE), Tris/Borate/EDTA (TBE) and Sodium borate (SB). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).
After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA bands. The ethidium bromide fluoresces reddish-orange in the presence of DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white (see figures).
Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ.
Typical materials and method
For an agarose gel electrophoresis, several items are needed:
There are several methods for preparing gels. A common example is shown here . Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a constant amount while length and breadth are varied as needed). The vast majority of agarose gels used in modern biochemistry and molecular biology are prepared and run horizontally.
After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. It is now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Agarose_gel_electrophoresis". A list of authors is available in Wikipedia.|