Methods and reagents: Degraded DNA and gel tornados 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 a case of inexplicable DNA degradation and tornados seen in agarose gels. For details on how to partake in the newsgroup, see the accompanying box. Degraded DNA ************ Some people have the worst luck when it comes to running gels. Recently, Caroline Jones (caroline@fcrd.gov.uk) wrote about an inexplicable case of DNA degradation that was occurring in one of two identical horizontal agarose gel electrophoresis units run side-by-side. After pipeting aliquots of PCR-amplified DNA into two identical 1.2% agarose gels cast in 1 x TAE buffer and placed in similar electrophoresis units, one unit produced a gel with all samples looking like the DNA had been degraded by nucleases when stained with ethidium bromide and viewed under UV light. The other gel unit run simultaneously and under the same conditions (3 hr at 90 V) produced a gel with normal looking intact DNA bands. The apparent degradation observed in the first unit worsened over time and progressively spread up from the anode into the gel during subsequent runs. Oddly, when the gel tray from the good unit was transferred into the bad one and a gel run, the result was degraded DNA. However, when the tray was returned to the good unit again, another gel run showed the DNA degraded. Being quite baffled, this confused researcher turned to the methods group for help in solving the mystery. Netters coming to the rescue proposed that one of the units was having an electrical problem, or that the gel had not been given enough time to solidify. Incomplete setting of the gel was thought to prevent the DNA from being resolved into distinct bands. However, this could not explain why the strange result happened in only one of the gel units. Differences in electrical sources, on the other hand, could not be excluded. Because the molecular weight markers run along with the samples also looked degraded on the same gel, contamination with some microorganism that might be secreting a nuclease was suspected. The DNA and all the sample reagents were eliminated as the source of the contamination because the same samples ran prefectly in the good unit when placed next to the bad one. To rid the bad unit of the suspected contaminant, the buffer tank was rinsed with Decon-90 and both trays were subjected to both 5% HCl and 0.5 M NaOH. However, after all the cleaning up, there was still no improvement in gels run in the bad unit. One person thought that perhaps the problem was due to traces of Fe2+ ions that might be found within certain batches of EDTA, or from other ions that could be seeping into the distilled water supply. He pointed out a study [1] that showed specific degradation of Streptomyces plasmid DNA during electrophoresis in TAE buffer. After careful consideration of all factors and numerous experiments, that study proposed that the electrophoresis process itself could be generating an unknown agent through the creation of molecular oxygen at the anode, which might interact with an EDTA-Fe2+ complex, and that this is what might be attacking the Streptomyces DNA. However strange, the phenomenon seems reminiscent of the glowing blue gels seen by netters when TAE buffer has gone bad (see TiBS 19, 556-557). Unfortunately, in the particular case described here, the exact source of the degradation was never pinpointed. We're not in Kansas any more **************************** Recently, one netter had difficulties in loading samples into the wells of an agarose gel. The DNA would not stay at the bottom, but instead, floated out of wells and diffused into the buffer causing a vortex of loading dye that resembled miniature blue tornados. As this person had purified his DNA using a Qiagen mini-prep kit, netters immediately assumed that residual ethanol from the final wash solution could be left in the samples. If any ethanol had remained after the purification, decreased density of the loading solution could cause the DNA to float. Most people suggested that every last bit of ethanol be removed from the samples by drying them in a roto-vac. Netters say that this problem is quite common and that any residual buffer components or organic compounds including ethanol, isopropanol, sodium dodecyl sulfate, mineral oil or phenol in DNA samples causes the loading solution to come out of the wells. In addition, the problem is more likely to occur with DNA samples produced by the quicker plasmid prep methods and by people who tend to skip precipitation and drying steps to save time. Others thought that perhaps the loading dye solution containing glycerol or ficoll had been contaminated and that the polymeric sugars had been degraded causing changes in density. One person had a similar problem in loading, but suspected that it was owing to changes in the density of the running buffer. Netters typically re-use running buffers several times, and adjust for evaporation by topping up the volume with fresh buffer. They say that the buffer can be left in the chamber for weeks or months and can be used up to more than ten times before needing replacement. After that, it is generally felt that the buffer has hit the limit and should be replaced because the accumulation of salts changes the density of the buffer and this is what causes the tornados. If left for a long time, however, there is a higher risk of contamination or changes in salt concentrations causing ionic differences between the gel and the buffer. For this netter, as with many problems with electrophoresis, the cure for the tornados was simply to click his heels three times and change over to a freshly prepared buffer solution. So Sensitive ************ When cloning into a plasmid vector that does not have an interruptible lacZ gene for blue/white screening of recombinants, it would be advantageous to screen bacterial colonies for sensitivity to an antibiotic. For example, when cloning in the older vectors, such as pBR322, which has both tetracycline- and ampicillin-resistance genes for selection, no indicator for insertional inactivation is available. Therefore, screening for inserts must be done by either toothpicking colonies or replica-plating them onto separate plates using velvet pads, which can add several days to the prep time of media and the manipulation of colonies. Recently, one netter was looking for a method to select for one antibiotic- resistance gene and screen for the loss of the other on the same plate. Cloning gurus in the methods group are familiar with two techniques for doing this. For the selection of tetracycline-sensitive bacteria [2], a specialized low-nutrient media containing chlortetracycline and fusaric acid can be used to kill cells that are resistant to tetracycline. The chlortetracycline induces expression of tetracycline resistance, which causes cell membrane modifications, making the cell hypersensitive to fusaric acid, a lipophilic chelating agent that is thought to disrupt the membrane [3]. While only 90% as efficient as actually streaking out the bacteria, the selection can save tremendous amounts of time. Another technique used in screening for ampicillin sensitivity is to flood starch-containing plates with colonies growing on them with a solution of penicillin-G and iodine. The beta-lactamase encoded by ampicillin-resistant bacteria cleaves the beta-lactam ring of penicillin to produce penicilloic acid, which binds iodine, thus eliminating the starch-iodine complex from the colonies. Colonies resistant to ampicillin therefore appear to clear, while sensitive ones remain colored brown [4]. Between the Sheets ****************** Researchers who perform multiple hybridizations within the same bottle when using a bottle and rotisserie oven system, pack pieces of fine nylon mesh between the membranes to separate them from each other and the glass bottle. Netters have complained that the replacement nylon mesh sold by companies such as Hybaid is far too expensive. As an alternative source of cheaper supplies, they say that plastic screen mesh for windows and doors purchased from a local department store or polyester mesh typically used for silk-screening, which is available at a crafts shop, can be used as replacement for the original hybridization netting without any troubles. References ********** [1] Zhou, X. et al. (1988) Nucleic Acids Res. 16, 4341-4352 [2] Maloy, S. R. and Nunn, W. D. (1981) J. Bacteriol. 145, 1110-1112 [3] Bochner, B. R. et al. (1980) J. Bacteriol. 143, 926-933 [4] Boyko, W. L. and Ganschow, R. E. (1982) Anal. Biochem 122, 85-88 ******************************************************************************* 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/apr97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: Degraded DNA and gel tornados. Trends in Biochemical Sciences 22(4):182-183. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************