Question

How is Agarose gel electrophoresis done?

Answer 1

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size.

To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore, when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size on an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along.

The rate of migration of a DNA molecule through a gel is determined by the following:

1. size of DNA molecule;
2. agarose concentration;
3. DNA conformation;
4. voltage applied,
5. presence of ethidium bromide,
6. type of agarose and
7. electrophoresis buffer.
After separation, the DNA molecules can be visualized under UV light after staining with an appropriate dye.