by Alex Powell

Created on: March 31, 2009   Last Updated: April 02, 2009

Electrophoresis is the movement of charged particles in an electrical field, first characterised in solutions by F.F. Reuss. In short, the higher the voltage, the lower the viscosity of the medium; the smaller the charged particle and the greater the charge on that particle the faster it will move. This is used in microbiology to determine the size of unknown particles by making all variables constant, so that the size becomes the sole determining factor in how fast the particle moves.

In gel electrophoresis a solution of large biological molecules (DNA, RNA or protein of unknown size and/or composition) that have been specially prepared is injected into a small well on a gel 'plate'. On the application of an electical field the particles will migrate through gel and become seperated by size; smaller molecules will move faster through the gel. The unknown solution is 'run' with a solution or solutions containing particles of a known size. This allows comparison and primary analysis of the size of the unknown particles.

This technique relies on the porous nature of the gels used, and requires that they be inert. The ability to control the size of the pores allows the gels to be applied to wide range of molecule sizes, and combined with different and variable voltages the technique can be pushed even further. There are two kinds of gel that are used, and both have very specific applications.

The first kind of gel used was polyacrylamide, which consists of long chains of acrylamide monomer combined with a crosslinking protein. This forms a very stable structure with the pore size controlled by the concentration in the gel. It was once the only gel available, but has been succeeded by agarose gel for most DNA anaylsis. Agarose consists of long molecules (polysaccharides) derived from seaweed which form a mesh with pore size dependent on concentration of agar polysaccharides in the gel.

Agarose gel is used to analyse DNA and RNA molecules. They are innately negatively charged and require only minimal preparation for analysis. They are treated with an alkali which disrupts the formation of complex structure, ensuring that their rate of travel is a product of their size, not their structure. They are analysed in a gel with concentration (pore size) suitable for the size of DNA fragment being analysed. For example, very short fragments will be resolved in 2% or even 3% gels, which cause very slow but very clear migration , reffered to as good resolution. 0.7% is the lowest

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