Methods and reagents: Glowing blue gels and nuked nucleases 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 unidentified blue fluorescence observed emanating from agarose gels, and the use of microwave ovens for DNA restriction digests. For details on how to partake in the newsgroup, see the accompanying box. Netters have observed two types of blue fluorescence on agarose gels stained with ethidium bromide and observed under UV illumination. In the first case, bright blue spots were seen wherever the gel had been touched with a bare hand. However, when a black and white polaroid snapshot was taken with an orange Wratten filter, the spots were invisible. Hypotheses about the source of the blue spots focused on the transfer of fluorescent substances, such as the talcum powder used to lubricate vinyl or latex gloves, and skin oils, from the hands onto the gel. In the second case, one netter had observed an entire agarose gel in Tris-acetate-EDTA (TAE) running buffer glowing bright blue under UV, which made it very difficult to see the orange ethidium-bromide-stained DNA bands. The phenomenon seems to be specific for this buffer, since it doesn't occur in Tris-borate-EDTA (TBE) buffers. One explanation was that the glow results from contaminants within the agarose that may not be apparent when the gel is made at normal concentrations of 0.8-1.2% agarose (w/v), but that higher percentage gels become autofluorescent; alternatively, overheating the molten agarose in a microwave oven might cause it to degrade into autofluorescent monomers. It became apparent that the source of both the spots and the completely blue gel might be the same, since over-handling of the gel with contaminated hands could cause the whole gel to glow. Some people speculated that the source of the blue glow could be phosphorous-containing fluorescent whiteners from laundry detergents, which could be transferred to your hands by wiping them on clothing. Touching the gel might then transfer the dye directly onto it. Another idea emerged when glowing gels were reported by two other netters who were using the same TAE buffer composed of 40 mM Tris, 20 mM acetate, 2 mM EDTA, pH 7.8. It was noticed that the glow occurred only when an older stock of 50x concentrated buffer was used. John Nash (nash@nrcbsa.bio.nrc.ca) wrote that glowing gels were seen when run at high voltage for a very short time, or if allowed to run overnight at low voltage without recirculating the buffer. Interestingly, while the gel was running under UV light, a blue glow was seen creeping up from the bottom of the gel over time, and any DNA in its path was degraded. This led him to think that the buffer in the positive ion chamber was becoming more acidic, and that perhaps this change in pH or a breakdown product of the acetate buffer degraded the DNA. Another netter, Scott Mulrooney (scott.mulrooney@um.cc.umich.edu), confirmed this and noticed that buffer that is more than 2-4 weeks old sometimes contains a visible mold adhering to the inside of the bottle, and that freshly prepared buffer never causes the glowing. The bright blue glow of a six-month-old TAE buffer could not be eliminated by extraction with butanol or passage through a cation-exchange column, but could be removed with activated charcoal. It therefore seems reasonable that buffer breakdown or modification, or perhaps a bloom of fluorescent microorganisms that can degrade DNA by secreting nucleases, may cause the blue glow. Alternatively, it could be caused by a fluoroacetate derivative or some other precipitate that forms as a result of washing the gel with fluorinated tap water. In any case, the common factor seems to be the use of old solutions, and may be corrected by preparing fresh buffer stocks. Microwave restriction digests ***************************** Recently, there was a discussion about using microwave ovens to speed up restriction digests. This is accomplished by exposing a DNA sample, pre-mixed with enzyme and buffer, to microwaves for several pulses of 45 s interspersed with 2-3 s at room temperature. This method has been reported to give a 50- to 1100-fold decrease in the digestion time required for genomic DNA, and a 2.5- to tenfold decrease for plasmid DNA, when using a 700 W Amanda model R 311 T microwave oven [1]. One netter was curious as to why this would increase the apparent enzyme rather than heat-inactivating it, thinking that perhaps the enzyme was absorbing microwaves to increase activity. It was also thought that the DNA may be vibrating by resonance, thereby allowing more contact with the enzyme through increased Brownian motion. Unintuitively, aqueous solutions of DNA do not absorb 1-10 GHz microwaves (most household microwave ovens emit 2.45 GHz microwaves) any more than water does [2]. If the increase in activity is real, this might indicate that microwaves cause the enzyme to undergo structural changes. One netter was impressed that 2.5 ug of plasmid DNA incubated with 20 units of SacI and HindIII in a volume of 20 ul and microwaved in a 650 W oven for four pulses of 15 s followed by 2 min intervals at room temperature gave a completely digested product. Others were quite critical because the plasmid DNA would almost certainly have been digested within the same time without microwaving. They felt it unnecessary to use microwaves for restriction digests of plasmid DNA because the enzymes available from commercial suppliers are of such good quality and high activity that any plasmid DNA will be digested to completion within a few minutes, regardless of the microwaves. One person proved this by digesting plasmid DNA with EcoRI for 5 min under three sets of conditions: at 37 degrees C, at room temperature, or while being microwaved. All the samples were completely digested. Some were even more skeptical of the technique, stating that 20-50 ul of DNA solution would evaporate completely in a microwave on high for that time period. However, someone else wrote that when a microfuge tube containing this amount of water was tested, no evaporation occurred. The reason for this is unknown, but some unlikely explanations that the sample was smaller than the wavelength of the microwaves, or that the polypropylene tubes blocked the microwaves, were dismissed. Since genomic DNA samples generally require more time to be completely digested, and since digestion is dependent upon the quality of DNA, the method may find applicability for Southern blotting techniques such as fingerprinting, or restriction fragment length polymorphism analysis. However, caution should be taken when using this method for DNA samples to be used as template for Southern blots, since aberrant banding patterns have been reported. One study showed that conformational polymorphisms in bacteriophage lambda DNA were induced by microwaving at 2.45 GHz in a Sanyo model LM-2500LA oven for 2-8 s. After digestion with EcoRI and agarose gel electrophoresis, the changes in DNA migration patterns were presumed to be caused by bulges, bends or other distortions induced by microwaves. These did not appear to be the result of thermal transfer to the DNA because the exposure time was too short. The additional restriction bands were probably due to the enzyme recognizing either localized denatured regions or "pseudo"-restriction sites [3], while in another study it was shown that contaminating metal ions increase microwave-induced nicks in plasmid DNA [4]. However, using microwaves to denature a radiolabeled probe for use in a Southern blot may be beneficial. Denaturation of probe DNA using a long exposure (up to 5 min) to 2.45 GHz of microwave energy gave up to 20 times more sensitivity when detecting small amounts of immobilized DNA by Southern hybridization [5]. The reason for the increased sensitivity is unknown, but could be attributed to single- and double-stranded breaks incurred through microwaving and subsequent fragmentation by thermal denaturation, allowing the shorter probe DNA to diffuse more readily towards its target. While microwaving is claimed to decrease the time needed to digest genomic DNA samples [1], netters are not convinced that the microwaving of vector DNA is necessary, nor that the correct experiments have been done to demonstrate the microwave effects on restriction enzyme activity. In addition, the effects of such exposure on ligation and transformation efficiency have not been reported. References [1] Jhingan, A. K. (1992) Methods Mol. Cell. Biol. 3,270-274 [2] Gabriel, C. et al. (1987) Nature 328,145-146 [3] Narasimhan, V. and Huh, W. K. (1991) Biochemistry International 25,363-370 [4] Sagripanti, J-L., Swicord, M. L., and Davis, C. C. (1987) Radiation Res. 110,219-231 [5] Stroop, W. G. and Schaefer, D. C. (1989) Anal. Biochem. 182,222-225 ******************************************************************************* 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/dec94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - Glowing blue gels and nuked nucleases. Trends in Biochemical Sciences 19(12):556-557. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************