Gel electrophoresis

Gel electrophoresis is a method used to separate charged macromolecules of different sizes and estimate their length.

Gel electrophoresis is often used to separate PCR amplified DNA fragments:

The sample is loaded in small wells in one end of an agarose gel. An electric current is applied to the gel, with the positive electrode postioned at the end opposite the wells. The negatively charged DNA will move through the gel towards the positive electrode. Smaller DNA molecules move faster through the gel than larger DNA molecules, leading to size separation. After electrophoresis, DNA is visualized and appears as bands derived from DNA fragments of the same length. Only a large amount of DNA (millions of copies) can be visualized with this method. A “DNA ladder” containing a mixture of DNA fragments of known sizes is also loaded on the gel. The sizes of the DNA samples can be estimated by comparing the distance with the DNA ladder (Figure 1).

Figure 1. Fragments of different lengths from a PCR reaction are run on a gel. The fragments will move at a speed and a distance relative to their size: smaller DNA molecules will move in the gel faster and further than longer DNA molecules.

By mixing the PCR product with loading buffer, we can visualize how far the PCR product has traveled during gel electrophoresis. Loading buffer is also useful in making the sample heavier, so it will sink to the bottom of the gel.

There are many compositions for loading buffer, however, they typically contain the following chemicals:

• Coloring reagent, for example, xylene cyanol, cresol red, bromophenol blue, or orange G.

This will add color to the sample and help you visualize the sample position on the agarose gel.

• High viscosity reagent, for example, Ficoll, sucrose, or glycerol.

This will make the sample heavier by increasing the overall sample viscosity.

• Water to dilute the above mentioned reagents.