Agarose gel electrophoresis
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be purified from the gel if necessary.
The agarose gel is made by dissolving the solid agarose powder in the electrophoresis buffer. Usually Tris-AceticAcid-EDTA (“TAE“) buffer is used. A commonly used stock solution for TAE is 50 times concentrated (“50xTAE”; the standard procedure for preparation of 50xTAE is: mix deionised water with solid Tris powder, a certain amount of an EDTA stock solution, usually 0.5M EDTA pH8.0, and concentrated acetic acid to adjust the pH to 7.6). The agarose will only fully dissolve when boiled for a few minutes and the warm gel solution then is poured into a mold which is fitted with a well-forming comb. Cooling down to room temperature results in the slow formation of a solid gel when the concentration of agarose is between 0.5 and 2% (weight/volume).
To make the DNA visible in the gel, ethidium bromide is added to the gel solution and the buffer (it can also be left out of the gel and buffer; staining of the gel can be done in that case after the gel run..). This positively charged polycyclic aromatic compound binds to DNA by inserting itself between the basepairs (“intercalation“). The DNA bands can be seen by exposure of the gel to ultraviolet light, due to the the large increase in fluorescence of the ethidium bromide upon binding to the DNA.
Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. This buffer both conducts electric current and controls the pH of the solution during electrophoresis. DNA samples for loading into the wells (“slots”) of the gel are prepared by addition of a tracking dye (e.g. Orange G or Bromo Phenol Blue)which also contains a component (usually glycerol or sucrose) to increase the density of the sample to facilitate the loading.
Electrophoresis usually is at about 5 Volts per cm for 0.5 – 2 hours or more at room temperature, depending on the desired separation. Size markers may be co-electrophoresed with DNA samples, when appropriate for fragment size determination. Many commercial size-marker sets are available with different size ranges.
After electrophoresis, the gel is placed on a UV light box and a standard or digital photograph of the fluorescent ethidium bromide-stained DNA separation pattern is taken.
This is what a (black-and-white photograph of a EthBr-stained) typical gel looks like after the electrophoresis:
Above you see a black-and-white photograph of an agarose gel. Before we took this picture, we applied an electrical field across the gel which was submerged in a buffer solution. During this so-called electrophoresis the DNA fragments in the samples 1 to 11 moved from their origen, the sample wells (or: slots), through the gel towards the positive electrode that’s from top to bottom in the picture. The gel matrix acts as a sieve: smaller DNA molecules migrate faster than larger ones, so DNA molecules of different sizes separate into distinct bands during electrophoresis.
Plasmid DNA can exist in three conformations: supercoiled, open-circular (oc), and linear (supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc).
In vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell. In the laboratory, following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will sustain single-strand nicks. Given the presence of a break in only one of the strands, the DNA will remain circular, but the break will permit rotation around the phosphodiester backbone and the supercoils will be released.
A small, compact supercoiled knot of ccc-DNA sustains less friction against the agarose matrix than does a large, floppy open circle of oc-DNA. Therefore, for the same over-all size, supercoiled DNA runs faster than open-circular DNA. Linear DNA runs through a gel end first and thus sustains less friction than open-circular DNA, but more than supercoiled. Thus, an uncut plasmid produces two bands on a gel, representing the oc and ccc conformations. If the plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to a linear conformation.
Following isolation, spontaneous nicks accumulate as a plasmid prep ages. This can clearly be seen on gels as the proportion of the two conformations change over time: plasmids preps that have been thawed and refrozen many times, show more oc DNA than fresh preps.
This is a black-and-white photograph of an agarose gel containing ethidium bromide, after electrophoresis of three DNA samples. The gel was on a UV lamp when photographed. It was weakly orange while the DNA bands had a bright orange color due to the specific binding of the EthBr to the DNA molecules.
Note: the (linear DNA) bands in lane 1 cannot act as size markers for the circular DNAs in lane 2!!
See also SCLResources>Lambda DNA for information about the gel band pattern of a Lambda DNA digest.