1. Many workers do not like to include ethidium bromide in their gels and their running buffers, preferring instead to stain their gels after electrophoresis. It must be noted that ethidium bromide promotes the damage of DNA when viewed under UV light (by photonicking). For this reason, if the gel is run with ethidium bromide in the gel and the running buffer, it is best to limit the amount of viewing of the gel to prevent damage to the DNA molecule and subsequent smearing on reelectro-phoresis. However, with this limitation in mind, there is no noticeable difference between running the gel with and without ethidium bromide, providing that one system is solely used (i.e., either always run your gels with ethidium in the gel and running buffer, or never). The reason for this is that ethidium intercalation can affect the mobility of the DNA, especially where circular plasmids (either supercoiled or nicked circle) are concerned. However, if the presence or absence of ethidium is kept constant, then no difficulty is encountered. Note that ethidium bromide is both carcinogenic and mutagenic, and therefore must be handled with extreme caution.
2. The amount of DNA that can be visualized in a single band with ethidium bromide staining can, in ideal circumstances, be as low as 10 ng. In general circumstances, a fragment consisting of approx 100 ng of DNA will provide a well-visualized band. Note that these figures are for single bands. If a digestion produces a large number of bands, then relatively more DNA will have to be loaded to ensure that all bands are seen.
3. Loading buffer is a dense solution (usually either glycerol or Ficoll), which when mixed with a DNA solution (or restriction digest) gives the sample sufficient density to fall to the bottom of the sample well, which is already filled with running buffer. Loading buffers normally contain either one or two marker dyes, which migrate in the electric field in the same direction as the DNA. Two commonly used dyes are bromophenol blue and xylene cyanol. These dyes migrate at different rates from each other. In a 0.8% (w/v) agarose gel, bromophenol blue migrates with DNA of approx 1 kb. Xylene cyanol in the same gel migrates at approx 4 kb. These dyes are useful for monitoring the progress of an electrophoretic run and, thus, ensuring that the DNA does not pass out of the bottom of the gel.
4. When running an analytical gel, the optimal resolution is obtained at about 10 V/cm of gel. When fragments of 5 kb and above are to be analyzed, better resolution is obtained at about 5 V/cm. Fragments smaller than 1 kb are normally resolved better at higher V/cm.
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