Today's task is running gels, to see if our DNA extraction technique managed to extract DNA. Halfway through measuring out the agarose for the gel base I suddenly realised that I had very little idea of how these gels actually worked. Lab Rat job of the day therefore, was to find out more about them (hopefully in order to prevent further embarrassment of the NrdG kind).
Agarose gel electrophoresis is a basic method used to separate RNA and DNA of different sizes. The gel itself is made up of an agarose matrix (formed from agarose, ethidium bromide and TAE; Tris-acetate EDTA. More on those later) with little wells at the top to add the DNA. As DNA is negatively charged, you can run an electric charge across the gel causing all the DNA to migrate down it.
The agarose and TAE make up a matrix. The TAE acts as a buffer, keeping the agarose at a constant pH. The agarose makes the gel more difficult for the DNA to run through; which allows the DNA to separate. Small fragments can get through relatively easily while larger fragments take a lot longer.
Ethidium bromide is used to visualise the gels. It fluoresces under UV light when intercalated into DNA, allowing you to visualise any band containing ~20ng of DNA under a UV lamp. The problem with ethidium bromide is that it intercalates with any DNA, including the DNA on (say) your skin. This makes it a powerful carcinogen, so it's a good idea to wear gloves and coats while handling it.
As well as length, the distance moved by the DNA is also affected by its conformation. Linear fragments sort nice smart bands (ideally) while circular DNA just forms a sort of elongated blob. Because of this, circular DNA fragments (i.e from plasmids or viruses) are usually cut up using restriction enzymes. As we're just seeing if we've got any DNA at the moment, we haven't bothered cutting it up into pieces, we're just running the circular DNA from our viruses.
All fun stuff :)
Bioplastic from weaver's broom
1 day ago in Doc Madhattan