What Are the Steps in Gel Electrophoresis?
Gel electrophoresis is a process of separating bio molecules of different sizes by running them through a sievelike matrix using electricity. The larger molecules move more slowly, while smaller molecules slip through the matrix and move faster and farther, thus separating the different fragments based on size.
The first step to gel electrophoresis is to set the gel matrix. Agarose is used to separate DNA molecules, and acrylamide is used to separate proteins. The gel starts off as a liquid, which is poured into a molding tray. A comb is placed in the liquid matrix so that when the matrix solidifies, wells are formed to load samples in them. Once the gel has solidified, it is removed from the mold and placed in a special apparatus where current can be applied. A buffer that can act as a conductor of electricity is poured around the matrix.
The samples of bio molecules are usually mixed with a substance of high density (a viscous dye) so that they sink to the bottom of the well instead of floating away in the buffer. The dye also helps track the progress of the experiment. Each sample is loaded in a separate well. One of the wells is usually assigned for loading a marker, which has a set of fragments whose sizes are already known in order to allow for comparison with the samples being loaded.
When the current is switched on, the samples tend to move towards the positively charged side of the apparatus since the phosphate backbones of the molecules confer a negative charge on them. After the samples have run a sufficient distance, the matrix is studied to view the bands that are formed by the separation of the molecules.