Summary
Highlights
Agarose gel electrophoresis separates DNA fragments or other macromolecules by size and charge. The first step, gel casting, involves weighing agarose powder. The concentration of the gel (low for larger molecules, high for smaller ones) depends on the size of the molecules to be separated. The agarose is mixed with a buffer (like TAE or TBE) and heated until dissolved, then cooled to around 55 degrees Celsius.
While the agarose cools, an electrophoresis tank with positive and negative electrodes is prepared. A comb is placed to create wells for samples, and casting dams prevent the agarose solution from leaking. Before pouring, ethidium bromide is added to the cooled agarose. This highly toxic mutagen intercalates with DNA and fluoresces under UV light, making DNA bands visible. Handle it carefully in a fume hood.
After adding ethidium bromide, the agarose solution is poured into the tank and allowed to solidify at room temperature. Once solid, the comb and casting dams are carefully removed. The gel is then submerged in a running buffer (TAE or TBE) to provide ions for current and maintain pH. Next, DNA samples are prepared by adding a loading buffer containing glycerol and dyes like bromophenol blue. This buffer adds density, allows samples to sink, provides color for monitoring, and tracks separation progress.
Before loading samples, a DNA ladder (molecular-weight size marker with known fragment lengths) is loaded into the first well as a reference. Then, the DNA samples are loaded into the remaining wells. A lid is placed on the electrophoresis tank, and an electric current is applied. DNA molecules, being negatively charged due to phosphate groups, migrate towards the positive pole. Smaller fragments move faster through the gel matrix than larger ones.
Bromophenol blue, migrating faster than DNA, allows for optical control, indicating when to stop the electrophoresis before samples exit the gel. Once separation is complete, the gel is placed under UV light, and the DNA fragments appear as bright bands due to the ethidium bromide. Each band represents a group of DNA fragments of the same size. By comparing sample bands to the DNA ladder, the approximate sizes of the DNA fragments in the samples can be determined.