Appendix 3: Additional laboratory details
Lab 3: Agarose Gel Electrophoresis
Agarose is a highly purified form of agar that is extracted from seaweed. The density of the gel is determined by the weight percentage concentration of agarose in the solution. Different concentrations of agarose are used in gels depending on the relative sizes of the fragments expected or the separation desired. Acrylamide gels can be used when small pieces of DNA are to be resolved.
The molecular weight of DNA determines its rate of migration. Typically, the smaller the molecule, the faster it will migrate through the gel. However, DNA of the same size can have differing mobilities depending upon the shape: linear, circular relaxed, or circular supercoiled. Circular supercoiled DNA migrates faster than either linear or circular relaxed DNA, while linear DNA typically migrates faster than circular relaxed DNA.
When the voltage is low, migration of DNA is proportional to the strength of the electric field, regardless of the DNA size. At higher field strengths, larger molecules move disproportionately faster so that the resolution is decreased.
The buffer system can also affect mobility. If you forget to add the running buffer to the agarose gel, electrical conductance will be decreased and the DNA will migrate very slowly. Alternatively, if you use 10x or 50x buffer to make the gel, the ionic strength will be too high, and consequently the amount of heat produced will be excessive.
The sample is loaded after the gel is just covered with buffer. It is important to add just enough buffer to cover the gel, but not a lot more. Excess buffer allows a substantial conduction pathway on top of the gel instead of the desired pathway through the gel, so migration of DNA through the gel will be slowed.
Samples are mixed with a solution containing bromphenol blue and xylene cyanol as tracking dyes; the bromphenol blue migrates about the same rate as a 0.3-0.5 kb fragment and the xylene cyanol migrates similarly to a 4-5 kb fragment. This loading dye can also contain SDS, EDTA, and sucrose. The sucrose makes the sample denser than the buffer so the sample will sink to the bottom of the well when it is loaded. Generally 1-2 μL of loading dye is added per 10 μL of sample.
Ethidium Bromide Staining
Since DNA cannot be detected in a gel, ethidium bromide, a fluorescent dye, is added to the DNA. The planar, polycyclic ethidium bromide intercalates between the bases of double-stranded DNA and RNA. Ethidium absorbs UV light at 302 nm and produces a reddish-orange fluorescence at 590 nm; the amount of this fluorescence is proportional to the mass of the DNA, assuming there is enough ethidium present to saturate all binding sites on the double stranded nucleic acid. The fluorescence of RNA is less than that of an equal mass of DNA, because most of the RNA is single-stranded and less susceptible to ethidium intercalation.
Ethidium bromide can be added to the DNA in the gel in any of three way