Agarose Gel Electrophoresis of DNA
Preparing and Running Standard Agarose DNA Gels
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:
To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.
After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.
The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.
When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis.
Migration of DNA Fragments in Agarose
Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.
|Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.|
|Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are:|
|Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.
The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.
|Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).|
|Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.|
|Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.|
Agarose gels, as discussed above provide the most commonly-used means of isolating and purifying fragments of DNA, which is a prerequisite for building any type of recombinant DNA molecule.
By varying buffer composition and running conditions, the utility of agarose gels can be extended. Examples include:
Finally, if you haven’t had an opportunity to run agarose gels, try out the virtual agarose electrophoresis lab.
Advanced and Supplemental Topics