Agarose gel electrophoresis
Agarose gel electrophoresis is used for separating of large DNA fragments. Fragments are generally classed as large if they are between 100bp and 25kbp. These larger fragments require gels with larger pores, which is why agarose is used rather than standard acrylamide gels. Agarose is derived from seaweed and is made of repeated agarobiose (a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose.) The concentration of the agarose affects how easily the DNA can pass through it, and therefore the degree of migration. Distance travelled is also affected by the size of the molecule. Smaller molecules travel further as they experience less resistance than longer DNA fragments.
DNA has a fixed negative charge density, and so doesn't require treatment with SDS - sodium dodecyl sulphate. This is because DNA is already charge-independent, and its highly coiled double helix makes it shape-independent too. DNA samples tend to be treated before electrophoresis; the two most common processes being cutting it into smaller fragments using rectriction enzymes, and selection and amplification using the polymerase chain reaction. The DNA sample is mixed with a dye Ethidium bromide (EtBr) prior to it being inserted into the wells, to make it easier to see the bands of DNA. When this is exposed to UV light, it fluoresces.
The gel is used to prevent convection currents from dispersing the molecules - this causes the molecules to move as a band. The gel's pore size also controls the migration speed of macromolecules, acting as a molecular sieve. As the phosphate groups in the backbone of DNA are negatively charged, so when the molecules are pipetted into wells near the cathode the DNA frgaments will migrate to the anode when a charge is applied. DNA fragments move at different rates through the agarose gel depending on how small and compact they are. Then the size of the fragment can be ascertained by comparing the sample to a marker lane, containing fragments of known molecular weight or size.
- ↑ Voytas D, Agarose gel electrophoresis, Current protocols in molecular biology - edited by Frederick M. Ausubel, May 2001
- ↑ Smith DR, Agarose gel electrophoresis, Methods in Molecular Biology, 1993, 18: pp443-8
- ↑ Lee PY, Costumbrado J, Hsu CY and Kim YH, Agarose gel electrophoresis for the separation of DNA fragments, Journal of visualised experiments, April 2012, 20;62:3923