Methods and reagents: Agarose gel electrophoresis in your kitchen 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 describes how to make an electrophoresis system from common household items. For details on how to partake in the newsgroup, see the accompanying box. Home-grown electrophoresis ************************** Characterization of DNA molecules is usually done by electrophoretic separation through a gel matrix. Since some researchers are constantly trying to get away with the least expensive way to make their own equipment, and one thread of discussion this month surrounded the construction of an agarose gel electrophoresis box from scratch for under a dollar, this led me to recall some past discussions based on a similar premise - a low cost method of doing molecular biology in your kitchen or basement. Here I've compiled some of the ideas posted to the net for doing simple and cheap lab experiments at home, or as a demonstration in a high-school classroom. To construct a gel box for under a dollar, first you'll need a plastic box. A lid from an old molecular-biology-kit box, a toolbox, a fishing box, a pipet-tip rack, or a small (6 x 8 inches) Tupperware [TM] sandwich-saver will suffice. You'll also need a smaller box and a makeshift comb for casting the gel. The 2-3 mm wide plastic combs that come with disposable pre-cast gels are usually meant to be thrown away; however, they can be trimmed down with a razor blade and smoothed over with a nail file to the desired size and will probably last the lifetime of your homemade gel system. Electrodes can be made out of two paper clips, but the cathode will pit and eventually errode away to nothing after a few gel runs. Although it costs a little more, a better choice is a platinum wire, which can be purchased in various gauges and lengths from many scientific supply companies. To cut the cost, a cheaper nickel-chromium wire may be used as the anode since the anode will not pit n the same way as the cathode. Weird science ************* Chulho Kang (kang@msvax.mssm.edu) wrote that he has designed a gel box with carbon rods as the electrodes. For his system, two pencil cores act as the electrodes. To remove the core from a pencil, he flamed it with a bunsen burner to burn off the wood until only the core remained. He then made two holes a little larger than the carbon core diameter at opposite ends of a plastic box and pushed the pencil core through, allowing about 0.5 cm to stick out. The core was fixed in place with silicone aquarium sealer, and any leaks were plugged. He wrote that this type of electrode can be used for up to 100 gel runs before needing replacement. Alligator clips were used to connect the power supply; the pencil core was clamped; and the wires were rigged to a series of rechargable 9 V batteries. A similar system can be used as an inexpensive electroelution device or capillary gel system [see TIBS 19 (1994), 388-389 for references]. This can be set up easily, using a microtiter dish and a small capillary tube. To bend a capillary tube, heat it in a flame until it's pliable, then fold it to form a U-shaped structure. Fill the tube with electrophoresis buffer or molten agarose gel using a micropipet. Place the tube so that it spans two of the titer dish wells filled with electrophoresis buffer. An array of capillary tubes can be placed in the same dish so that several can be done at the same time. Tape a bent platinum wire into each well and connect them to your power supply with clips. The buffer can be TAE [40 mM Tris-acetate, 1 mM EDTA, pH 8.0] or TBE [89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.0]. The gel matrix can be made as 0.8-2.0 % w/v agarose, agar[1] or 10-20% Knox [R] unflavored gelatin, in 1x electrophoresis buffer. Test the system by transferring a dye solution containing standard DNA molecular weight markers before using it to separate DNA samples. Many food colors are anionic in TBE, and can be used as tracking dyes. The loading dye solution should be made more dense than electrophoesis buffer by mixing in 20% sucrose, 5% glycerol, or 10% Ficoll [R]. Various dye cocktails can be made by combining 0.05% w/v of any of the following: xylene cyanol FF (Sigma X-4126; cyan), bromphenol blue (Sigma B-5525; dark blue), orange G (Sigma O-3756; yellow or orange depending on concentration), or amaranth (Sigma A-1016; red). These dyes will migrate in the order listed here, with the red dye migrating the fastest, and will provide a pleasing visual confirmation that the system is operating properly. The dyes can also be used separately for color-coding samples. For example, I mix lambda-phage DNA digested with HindIII in with only the dark blue dye, a 1 kb DNA ladder with the cyan dye, and test samples with the red dye because it does not interfere with DNA fragments above 300 basepairs. The dyes can also be used as alternating colors to make sure you haven't added the same sample to the gel twice. The intensity of the colors can be customized by increasing the dye concentrations. One problem with orange G, however, is that it separates into two discrete bands, one that appears bright and one that blocks any DNA bands below 20 base pairs when the gel is stained with SYBR Green I and viewed under 254 nm UV light. Presumably, one component fluoresces and the other absorbs at this wavelength. For polymerase chain reaction (PCR) enthusiasts, cresol red (Sigma C-9877) will migrate at approximately the same distance as that of a 300 base pair DNA fragment in a 2% w/v gel, and tartrazine (FD&C yellow #5, Sigma T-0388) will migrate well ahead of this, about where PCR primers would be located on the gel. The advantage of using these dyes is that they can be added before the PCR and will not inhibit the amplification of DNA by Taq polymerase. [2] Make your own markers ********************* DNA molecular weight marker can be made for far less than those offered by biotechnology companies. The best known marker is lambda-phage DNA digested with HindIII, but other sizes of double-stranded DNA can be obtained by digesting with BstEII, EcoRI, StyI, or a combination of BstEII and ClaI. Netters feel that the best source of lambda-DNA (GenBank Accession No. J02459) is from an Escherichia coli lysogen carrying the lambda strain cI857tsSam7. After a temperature shift from 30 degrees C to 45 degrees C during exponential growth of the infected culture, virus particles are harvested to extract the DNA, which should yield approximately 5 mg of lambda DNA per liter of culture. [3] Alternatively, Bacillus SPP1-phage DNA (GenBank Accession No. X56064) digested with EcoRI will generate fragments varying by approximately 1 kb, ranging from 1 kb up to 8 kb. Another source of a 1 kb ladder is the yeast 2 micron DNA of 1019 base pairs (GenBank Accession No. J01347) cloned into pBR322 or a similar plasmid as concatamers. [4,5] For fractionating lower molecular weight DNA, pBR322 (GenBank Accession No. J01749) digested with HaeIII, and pUC19 (GenBank accession number M77789) digested with Sau3A, which gives separated bands between 78 and 1000 basepairs, are good substitutes. To visualize the DNA, the gel may be stained with ethidium bromide, SYBR Green I, silver stain, methylene blue, toluidine blue, azure A, brilliant cresyl blue or with any nonradioactive detection kit that uses a streptavidin-linked enzyme and biotinylated DNA. [see TIBS 19 (1994), 257-258 for a discussion of alternatives for staining gels]. Warning! ******** Although using a low-cost gel system can yield DNA pure enough for a cloning experiment, and a plastic gel box system could be made for less than a dollar, most netters would agree that it is probably not worth the time and effort spent in building one since a quality system can be bought for a few hundred dollars. If made in a slipshod manner, it could cost more in damages incurred to your house or lab if sparks were to set off a blaze. In addition, if you use it in a laboratory, it could get you in hot water with the occupational health and safety officials. The description of the electrophoesis unit here is not meant for unsupervised children and is recommended only for occasional use purely as an interesting or educational weekend project. It could be a dangerous undertaking and I will not take any responsibility for anyone who accidently electrocutes him-/herself with a pencil after reading this article. References [1] Bush, C. N. and Holmes D. S. (1982) Anal. Biochem. 119, 164-166 [2] Hoppe, B. L., Conti-Tronconi, B. M. and Horton, R. M. (1992) BioTechniques 12, 679-680 [3] Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) in Molecular Cloning: a Laboratory Manual, pp. 76-85, Cold Spring Harbor Laboratory Press [4] Hartley, J. L. and Donelson, J. E. (1980) Nature 286, 860-865 [5] Anonymous (1989) BRL Focus 11, 36 ******************************************************************************* 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/may95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Agarose gel electrophoresis in your kitchen. Trends in Biochemical Sciences 20(5):202-203. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************