Electrophoresis is a technique used to separate large molecules like proteins and nucleic acids based on their size. It relies on the principle that larger molecules take longer to migrate through a porous medium than smaller molecules.

Nucleic acids are composed of chains of nucleotides. The “backbone” of the nucleic acid structure is a repeating chain of phosphate groups and pentose sugars. At certain pH values, the oxygen atoms in the phosphate groups ionize, giving the molecule an overall negative charge. If exposed to an electric field, these molecules are attracted to the positive terminal.

DNA electrophoresis occurs through a gel composed of agarose, a compound derived from seaweed. This is immersed in a solution of a buffer (a substance which maintains a constant pH) which has the dual role of conducting electricity and ensuring that the DNA molecules are at a consistent pH to ensure ionization.

The gel is then placed in an electrophoresis tank loaded with buffer. Solutions containing the DNA are mixed with a blue dye (which travels before all of the DNA and shows us how far the samples have traveled) and loaded into wells in the gel. A power supply is connected, with the negative terminal to the loading end. The process is complete when the dye front has almost reached the positive end of the gel.

Gels are normally made containing a compound called Ethidium Bromide. This substance binds to DNA and fluoresces when exposed to ultraviolet light. Once the gel has run, it is photographed under UV light. DNA fragments of a similar size run together as “bands”.  Smaller fragments can penetrate further through the gel than larger fragments in the same time. This means that bands of smaller DNA fragments will be found closer to the positive terminal, while larger fragments will be found closer to the loading wells.

One well in a gel is always left for markers. This is a mixture of DNA fragments of known size which separate out into a ladder. We can estimate the size of the fragments we are investigating by comparing how far they have migrated with the migration of fragments of known size.

GENIE, located at The University of Leicester have prepared some excellent videos showing how to set up and run an agarose gel:

The Dolan DNA Learning Centre has a useful gel electrophoresis animation. (note : this opens to an external website).