Gel electrophoresis remains a cornerstone technique in molecular biology, enabling researchers to separate DNA fragments by size and charge for applications such as DNA fingerprinting, restriction mapping, and sequencing.
Because DNA itself is colorless, scientists add tracking dyes—blue or orange pigments—to the sample. These dyes migrate alongside the DNA, providing a visual reference that lets the user gauge progress and ensure accurate band identification.
In this method, a slab of agarose gel—a polysaccharide derived from seaweed—is prepared by mixing agarose powder with a buffer containing water and salt. The mixture is heated until the agarose dissolves, then cooled to form a porous matrix. The gel is placed in an electrophoresis chamber and covered with conductive buffer.
DNA samples, combined with a loading dye, are loaded into wells at the negative (−) end of the gel. A positive (+) electrode is positioned on the opposite side. The negatively charged phosphate backbone of DNA drives the fragments toward the positive electrode when the electric field is applied.
Smaller fragments encounter less resistance and travel faster, producing distinct bands that correspond to fragment size.
Loading dyes serve two primary functions: they increase the density of the sample so it sinks into the well, and they provide a visual cue that tracks the migration of DNA. Common dyes include bromophenol blue (optimal for fragments around 400 bp) and xylene cyanol (better suited for 2–8 kbp fragments). The dye is chosen so it does not react with or alter the DNA. Researchers typically use 5 µL of loading dye per 1 µL of DNA sample, following guidelines from NEB and Thermo Fisher.
Glycerol is added to the sample to raise its density. Without glycerol, the liquid sample would spread across the gel surface instead of settling neatly in the well, compromising the formation of clear, distinct bands.
For protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‑PAGE) replaces agarose. Bromophenol blue is routinely incorporated into the sample buffer; it travels at the same pace as the leading edge of the protein bands, offering a real‑time visual indicator of progress.
After electrophoresis, DNA‑binding dyes such as ethidium bromide are applied. Ethidium bromide intercalates between DNA bases and fluoresces brightly under ultraviolet light, making bands visible. Because it is mutagenic, strict safety precautions are required when handling and disposing of the dye.
