Methods and reagents: Slants and spots Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses some of the unexpected and sometimes unexplained blotches that may appear on your gels or film plates. For details on how to partake in the newsgroup, see the accompanying box. A new slant on agarose gels *************************** Ever have one of those days when nothing seems to go straight? Well, here's the slant on gels: problems with electrophoresis systems are common, and Christopher W. Botka (cwbotka@MERCURY.SFSU.EDU) was not only experiencing a bad hair day, but also had a horrible case of the slants - messy DNA bands that are skewed on an agarose gel, with the bottom of the ethidium-bromide-stained band appearing to migrate further than the top when the gel was viewed from the side. This phenomenon was happening to all samples on the gel, including the double- stranded DNA molecular weight standards. Since this was seen only occasionally, and was not dependent on significant changes in the protocol, this poor netter turned to the net for advice. Unlikely explanations ********************* One person thought that the reason for the slants was that the comb used for casting the gel was not inserted in a completely vertical position, thus causing the bottom portion of the sample to migrate slightly ahead of the top. However, this would have been quite obvious, since the wells would have slanted at exactly the same angle as the DNA bands. Another reason suggested is that, rather than allowing the molten agarose to cool slowly, Mr. Botka was forcing it to solidify too rapidly, perhaps by placing it in a cold room. The haste may have resulted in faster solidification of the agarose at the top, producing a higher concentration of agarose or buffer components at the top of the gel and therefore a tighter molecular sieve or a different ionic strength than at the bottom of the gel. A slower cooling process should then rectify the slants because it would create a more uniform concentration throughout the gel. However, since the gel setup time was not affecting the slants, this clearly was not the solution. Others thought that the problem was caused by differential heating of the gel during electrophoresis. The `inside' or `bottom' of the gel may become warmer than the surface during a longer run, causing the bands to migrate slightly faster than those within the cooler top portion of the gel. This effect may be particularly noticeable when agarose gels are run in the cold, at higher voltages, or when thicker gels are run with only a thin layer of buffer covering surface, which may not sufficiently diminish the heat produced. Another idea is that a loading solution that contains glycerol may cause differences in migration owing to changes in ionic conditions between the DNA solution and the gel. One way to avoid this would be to use a loading solution consisting of 3-10% ficoll in running buffer instead. Concentration gradients *********************** A simpler and more believable reason for the slants is that a different concentration of buffer was used within the gel than in the electrophoresis chamber, or maybe that the buffer was inadvertently omitted from the gel when prepared. Someone suggested that this can be avoided by immersing precast gels in running buffer for a few hours before use. In this way, a few gels can be poured at one time and allowed to sit within a tank of buffer for several days at 4 degrees C prior to running. One netter thought that the slants were due to the addition of ethidium bromide directly into the gel, and that this could cause strange migration patterns. For example, running the gel with ethidium bromide added to the running buffer and a different concentration of ethidium bromide in the gel could cause the bands to shift as the dye diffuses into or out of the gel during electrophoresis. It is also possible that the effect is exaggerated when the gel is stained after the run and allowed to destain for a long time. This could cause the diffusion of molecules out of the matrix, pulling the DNA bands with them, so that they tend to slant away from the center of the gel. If this were the case, however, the bands would appear to slant in different directions, depending on their location in the gel. Although the exact reason for Mr. Botka's problem was not apparent, many factors could have contributed to the abnormally slanted DNA bands. In this case, the source of the problem was found to be inconsistancies between the gel and the running buffer. Since boiling led to evaporation of up to 10% of the water from 80 ml volume of agarose solution, the slants were eventually corrected by covering the container while the solution was being boiled. The amount of water that had evaporated was determined by weight and added back to make up for the loss before casting the gel. Spots: A case of Northern exposure ********************************** Allison Haggarty (mdah@musica.mcgill.ca) was performing some northern hybridizations when she found that some of her blots, which had been hybridized with a 32-P-labeled probe and exposed to X-ray film for 24 hours, looked as if they had come down with a horrible case of the measles. Spots ranging from two to 12 mm in diameter were randomly spread across the x-ray film. Andre Hamel (hamel@cc.umanitoba.ca) suggested that the random spots were due to the presence of unincorporated label during hybridization, which could happen if the radioactive nucleotides were not sufficiently separated from the labeled probe before being added to the hybridization mixture. Several methods used to remove the labeled probe from the nucleotides were discussed, including a spin column technique using Sephadex G-50, a commercial centrifuge unit with a 50-100 kDa molecular weight cutoff, and differential precipitation of the higher molecular weight probe. However, since the random spots were seen throughout the X-ray film, and no background was seen over the area that had been exposed to the membrane, it was thought that something else was causing the measles. Dr Haggarty didn't think the spots were the result of static electricity discharges from the plastic wrap, because they covered a wider area than the wrapped filter membrane and did not resemble lightning shocks. Instead, she had some suspicion that the talcum powder from the latex gloves worn during the experiment may have contaminated the film. It is not known why this would expose the film in this way unless the powder is very hygroscopic, causing water droplets to aggregate over the powder before the X-ray film was placed on top. Any exposure of X-ray film to water before development leads to blackening of the film when it is developed. One netter wrote that a bad lot of charged nylon membranes may have been the cause, since he had encountered the same problem when performing Southern hydridizations using a particular brand of membrane. Someone else thought that the radiography cassette may have been contaminated or placed very close to another one that had a colony- or plaque-library- screening hybridization on it. Radiation coming through a cassette has been found to expose X-ray film within another placed back to back with it in the same freezer. This would also explain why the spots appeared everywhere on the film. Unfortunately, the exact source of the measles has not yet been determined and may remain a mystery for some time. The good news is that further exposures using a different set of film plates from the same box but within a different cassette gave clean results. ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jul94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - Slants and spots. Trends in Biochemical Sciences 19(7):300-301. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************