Field Inversion Agarose Gel Electrophoresis of DNA David L. Weller and Peter A. Gariepy Department of Agricultural Biochemistry. Unlverslty of Vermont, Burlington, VT 05405 Laboratory courses in biochemistry and molecular biology may include an exercise on agarose gel electrophoresis of DNA to acquaint students with this widely used method of senaration and characterization. Examples of such exercises may be found in the literature cited ( 1 , 2 ) . The development of pulsed-field electrophoresis has made i t possible to separate larger DNA molecules than was previously possible using agarose pel electrophoresis (3).A laboratory experiment is-deicribed that can be used to introduce students to pulsed-field electrophoresis of DNA as represented by field inversion (agarose) gel electrophoresis (FIGE) (4). The enhanced separational capability is seen by comparing the FIGE results with those of conventional (continuous field) agarose gel electrophoresis. Carle e t al. (4) showed that by periodically inverting the electric field in one dimension, the range of fractionation of DNA molecules in agarose gels could be extended to greater than 700 kilobase pairs (kb). FIGE, as this technique is referred to, uses the same electrode arrangement (i.e., apparatus) as conventional agarose gel electrophoresis. Net mieration is achieved using this procedure by running for a longer time in the forward direction than in-the backdirection, and a window of fractionation is ohtained for differentsized DNA molecules by varying the switching cycle (4). Additional expense associated with the FIGE procedure when compared with conventional agarose gel electrophoresis comes from the need to reverse the polarity of the dc power supply periodically. For the experiment described the relatively inexpensive unit of Larson e t al. (5) was used to Meihods and Materlals Lyophilized Lambda DNA from E. coli strains GM 119 and W3110 and Xho I restriction endonuclease were ohtained from Sigma (St. Louis). The DNA was reconstructed with distilled water to give a concentration of about 0.5 pg1eL. One microliter of the Xho I solution was diluted just prior to use with 13 pL of a solution that contained 6 mM TRIS-HCI, pH 7.9,6 mM MgCIz, 150 mM NaCI, 6 mM 2-mercaptoethanol tsolution A), giving a working solut~onthat had 1 unitl#L of activity. Two and one half micrograms of Lambda treated for 1 hat 3 7 T wnh 5 units ofXho I.The DNA in 5 -I)NA - - - war ~~pL was diluted with 115 pL of solution A in a 1.5-mL micracentrifuge tube, and 5 pL of the restriction enzyme was then added. Following the incubation, the DNA was precipitated by adding 12.5 pL of cold 3 M ammonium acetate and 323 eL of cold 95%ethanol, mixing, and storing the sample for 20 min at -70PC or overnight at -20% The precipitate was collected hy centrifuging the sample in a microcentrifuge (Eppendorf,VWR, Rochester) at 16,000g for 30 min in the cold, and the supernatant was discarded. Next 10-20 pL of eold 70%ethanol was gently layered on top of the sediment and then decanted by placillg the tube upside down for a few minutes. This was repeated with cold 100%ethanol. The air-dried precipitate was dissolved in 50 pL of a solution which contained 1mM TRISHCL, pH 7.5, 1 mM NaCI, 1mM Na2EDTA, 9% glycerol, 0.25% hromphenol blue, and 0.25% xylene cyanote FF (solution B). Ten microliters of the digested DNA and an equal volume of undigested Lambda DNA were lbaded into separate wells in the agarose gel for electrophoresis. A simpler procedure also has heen used to obtain Xho I digested DNA for electrooharesis. It differs from that deacribd above mainly in that the DNA was not precipitated idlowing Xho I treatment. Two and me-half micrograms of DNA war treated with 5 unita of Xho I in 50 pL of solution A and then 5 aL of a solution containing 10mM TRIS-HCI, pH 7.5,10 mM NaCI, 10mM ~~~~~~~~~
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NaZEDTA, 2%sodium dodecylsulfate,and 50%glycerol (solution C) was added. Ten microliters was analyzed eleetrophoretically. The buffer for electrophoresiscontained in aliter of solution 10.78 g of TRIS, 5.5 g of boric acid, and 0.74 g of NazEDTA (solution Dl. Electrophoresis was in 0.8% agarose (DNA grade, Bio-Rad, Richmond, CA) containing solution D using a submerged horizontal slab apparatus (Mini-SubCell, Bio-Rad) that was placed in a bench-top cold box set at 4 C ( 6 ) . The field strength was 10 Vlcm. Field inversion employingthe unit of Larson et al. (5) attached to a 500-V dc power supply (Gelman,Ann Arbor) was for 20 h at 0.9 s forward and 0.55 or 0.45 s hack. The conventionalagarose gel electrophoresis run was stopped when the bromphenol blue had migrated about 4 em into the gel. Temperature of the electrophoresis buffer was 8 to 10 'C. Gels were stained with ethidium bromide, and the DNA bands were observed under ultraviolet light. Results were recorded by photographing the gels. Gels and solutions containing ethidium bromide were decontaminated as described by Lunn and Sansone (7). Results and DISCulislO~ Xho I cleaves Lambda DNA (48.5 kb) a t one site ( 8 ) producing fragments of 33.5 kb and 15 kb (4, 5). Lambda DNA and the Xho I fragments are readily separable by FIGE (Fig. 1). This was clear when the three different-sized DNA moiecules were present in the same sample (Fig. 1, lanes 3 and 7). Conventional agarose gel electrophoresis separated
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Flgure 1. FIGE of DNA. Samples were lmded in wells correspondingto lanes 1. and 7 at 0. LBrnWa DNA from E. coll host strain GM 119 (lane I) and W3110(lane5)digestedwilh Xho I, lanes 3 and 7, respectively. Theswitching cycle was 0.9 s forward and 0.55 s back. Forward was toward the positive pole, which corresponds to the bottom of the photograph. 3. 5.
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Number 1 Januaw 1991
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cules than did conventional agarose gel electrophoresis (Figs. 1and 2). The FIGE results (Fig. 1)are similar to those reported for Lamhda DNA and the Xho I fragments by Carle e t ~ a l .(4) and Larson et al. (5).The results (Fig. 2) are consistent with those of Carle et al. (4) who reported good fractionation of DNA molecules up to 15 kb and slight fractionation in the range 15 to 100 kb on conventional agarose eel electroohoresis. Xho I c~kevedtheLambda DNA from strains GM ll9and W3110 of E. coli (as indicated hv the suoolier and Fie. 1). Thus either one or both of the pieparati& can be used in the laboratory experiment described. Ethidium bromide can be included in the agarose gel and electrophoresis buffer (1,2). The advantage of this is that the gel can be examined directly under UV light during and at the end of the run. Precautions should be taken when using ethidium bromide, for it is a mutagen (1,2). Protective gloves should he worn when using solutions of the compound and handling gels that have been stained with it. Safety glasses should be worn when observing gels illuminated with UV light.
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Acknowledgment
I wish to thank graduate assistant Dosinda Huerata for her help with the FIGE exercise.
Figwe 2. Conventional agarose gel electrophoresis of DNA. The same s a m ples and in me same order as in Flgure 1.
1. ~ l ~ - d ~ .R. . R.;~ ~ i n i t hJ.sM.; , wiikinaon,~. wiley: N e w Yark, 1985:pp99-101. 2. Boyer. R. F. Modern Erperirnenlol Biochernialry; Addison-Wdey:Rending, MA,
the Xho I fragments (Fig. 2, lanes 3 and 7) but would not separate the Lamhda DNA (48.5 kb) and the larger Xho I fragment (33.5 kh) to any great extent (Fig. 2, lanes 1,3 and 5,7). FIGE clearly gave a better separation of the DNAmole-
7. Lunn,GI; ~ & a o n e , B.Anol. ~. Biorhorn. 1987,162.45M58. 8. Ginger-. T.R.; Myera. P. A.; Olson, J. A,; Hanberg. F. A,; Rebuts. R.J. J Mol. Biol. 1978,118,llb122.
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