Agarose gel electrophoresis

Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length.

Related Topics:
Molecular biology - DNA

~ ~ ~ ~ ~ ~ ~ ~ ~ ~

It operates by a mechanism similar to sifting molecules through a sieve; an electric field is used to drag negatively charged DNA molecules through a gel matrix, and the shorter DNA molecules move faster than the longer ones since they are able to slip through the gel more easily. Proteins can also be separated due to different charges and sizes. It can be used for the separation of DNA fragments of 50bp up to several megabases. Large DNA molecules are only able to move end on in a process called "reptation" and are more difficult to separate. In general the lower the concentration of agarose, the larger is the ideal size of a molecule to be resolved up to 750Kb. The disadvantage of lower concentrations is the long run times (sometimes days) and the problem of handling the fragile gel.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~

The rate of migration is affected by a number of factors. The concentration of agarose is one that has been mentioned. The conformation of DNA is also a factor and is demonstrated by the three forms of a plasmid: superhelical, nicked, and linear. Each form runs differently, the superhelical the fastest and the linear form the slowest. The presence of ethidium bromide (EtBr) in the gel causes DNA to run slower due to EtBr's ability to intercalate and uncoil DNA. The voltage is also a factor in migration and can only be so high before a decrease in resolution (~5-8 V/cm). Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Bromphenol Blue and Xylene cyanol FF are used and run at about 300bp and 4kb respectively.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~

There are a number of buffers used for agarose electrophoresis, but only two will be mentioned here: tris acetate EDTA (TAE), and sodium boride (SB). TAE has the lowest buffering capacity but provides the best resolution. This means a lower voltage and more time, but a better product. SB is relatively new and is ineffective in resolving fragments larger than 5kb but it has the highest buffering capacity allowing voltages up to 350V{{fn|5}},{{fn|6}}.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~