By Dianne Hermance, Updated Aug 30, 2022
Gel electrophoresis is a cornerstone technique in molecular biology that separates nucleic acids and proteins based on size and charge. By applying an electric field across a gel matrix, charged molecules migrate at rates inversely proportional to their length, allowing precise size estimation.
The most common medium is agarose, a purified polysaccharide derived from seaweed. Agarose forms a porous network that permits molecules of different sizes to traverse at distinct speeds. For fragments smaller than ~200 bp, a polyacrylamide gel offers higher resolution, though it requires careful handling due to its neurotoxic properties.
Before pouring, the gel solution is mixed with an intercalating dye—traditionally ethidium bromide (EtBr)—which fluoresces under UV illumination when bound to DNA. Modern protocols increasingly employ safer alternatives such as SYBR Safe or GelRed, albeit at a higher cost.
During gel casting, a comb creates wells that are later filled with sample mixed with a loading dye. The dye tracks the progress of the sample and adds density to prevent diffusion.
Once the gel solidifies, it is submerged in a compatible running buffer. The wells are loaded with the DNA or protein samples and a DNA ladder—a set of fragments with known sizes— is placed in a reference well. A constant voltage (typically 80–120 V) drives the negatively charged molecules toward the positive electrode.
After electrophoresis, the gel is exposed to a UV transilluminator. Bands corresponding to DNA or RNA fluoresce; protein bands may be visualized using Coomassie or silver staining. The migration distance of each band is compared to the ladder to deduce fragment sizes, while band intensity indicates relative concentration.
Careful handling of EtBr or its alternatives is essential, as these compounds can intercalate into DNA and, in the case of EtBr, are considered mutagenic.
High‑quality results depend on meticulous gel preparation: using freshly prepared, RNase‑free buffers; ensuring uniform gel thickness; and avoiding cross‑contamination between samples. Clean, well‑resolved bands free of smearing indicate successful separation and accurate downstream analyses.
Gel electrophoresis underpins a wide range of applications—from DNA fingerprinting in forensic science to assessing PCR products, cloning fragments, and verifying protein purity. Reliable interpretation of electrophoretic data is thus critical for both research and clinical diagnostics.
