Gel electrophoresis is used in a range of applications, including DNA fingerprinting, a profiling technique used to establish identity; the isolation and removal of DNA fragments of interest; and the identification of mutations linked to disease genes. The volume of gel needed for electrophoresis is determined by the size of the casting tray. Gel density—the percentage of solute molecules (agarose) to solvent (buffer)—determines how fast the DNA will migrate. The greater the gel density, the slower the rate of migration. Typically, gels are poured in a range of 1 to 3 percent agarose by mass. Lower concentrations are generally better when samples contain longer DNA because larger molecules do not move as easily through the gel, and this allows for greater separation between bands.

When pouring an agarose gel, as well as when running a gel, follow a careful protocol. Be mindful of when and how errors may occur. Bring the gel solution to a rolling boil in its swirl bottle or flask on a hot plate or in a microwave. If the powdered agarose is not fully dissolved, a finished gel will have regions of different concentrations, and the DNA samples will not separate correctly. Allow the solution to cool after boiling. Otherwise, it may damage the gel casting tray when poured. If the agarose cools too much and starts to solidify before pouring, reheat and recool it.

Once the solution has sufficiently cooled, make sure the casting tray is on a flat and level surface to obtain uniform thickness when the gel solidifies. Pour the solution, and look for air bubbles. If you find any, remove them immediately. As the solution cools further, sugar polymers cross-link with each other, and the gel begins to solidify. It's important to leave the gel undisturbed for at least 20 minutes for this to occur.

When loading a hardened gel into the gel box of the electrophoresis apparatus, position the wells near the negative electrode, or black end. Failure to do so will cause the DNA to migrate in the wrong direction when electricity is applied. When loading the wells with DNA, place the pipette's tip directly over each well, but not so close as to puncture it. Once electrical leads are plugged into the appropriate plugs—red to red, and black to black—it's time to run the gel. Running a marker, or control mixture of fragments of known size, with every gel will help with troubleshooting. For example, poor separation between bands may be the result of not having run the gel long enough, while lack of DNA bands may be the result of insufficient DNA being loaded or improper loading.

Throughout the process, adhere to proper techniques and safety rules. When handling a hot flask, always use hot pads. When handling gels, be aware that low-percentage gels can be especially weak, and high-percentage gels especially brittle. When staining gels, cover the workspace and wear gloves and protective goggles. Ethidium bromide, the most commonly used DNA stain, is a mutagen and suspected carcinogen. Be sure to follow hazardous waste disposal protocols.

Print Background Essay