Method to reduce the quantity of ethidium bromide required to stain DNA in agarose gels.

Ethidium bromide-agarose gel electrophoresis is an extensively used procedure for the analysis of DNA; it has been reported to be sensitive to as little as 50 ng of DNA (7). The standard methods for staining gels involve adding 0.5 μg/mL ethidium bromide to both the agarose gel and the running buffer or soaking the gel in a solution of ethidium bromide after electrophoresis has been performed (6). Intercalation of the ethidium bromide molecule between DNA bases causes fluorescence under ultraviolet (UV) light (6). Although fluorescence is much greater for the DNA-ethidium bromide complex, unbound ethidium bromide is responsible for the background fluorescence sometimes seen on agarose gels (6). Ethidium bromide is a suspected carcinogen and a known mutagen (1,4). Time-consuming deactivation procedures are therefore necessary before disposal of this substance (2,3,5). We describe a simple method that reduces the quantity of ethidium bromide necessary to stain DNA in agarose gels. This modified procedure consists of adding 0.25 μg/mL ethidium bromide to the agarose gel only and then performing electrophoresis as usual. To demonstrate the sensitivity of this modified staining procedure, a series of bacteriophage λ DNA (Sigma Chemical, Poole, England, UK) dilutions (16–500 ng) were prepared in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and mixed with an appropriate volume of 6× loading buffer (4% sucrose, 0.25% bromophenol blue). DNA samples were then electrophoresed on submarine 1% agarose gels in TBE running buffer (90 mM Tris-HCl, 90 mM boric acid, 1 mM EDTA, pH 8.0) at 70 V for 2 h. Gels were photographed on a 302-nm UV light transilluminator after staining with either the standard method or our modified procedure. Figure 1 shows the typical data obtained with each protocol. The results when 0.5 μg/mL ethidium bromide was added to both the gel and the running buffer are shown in Panel A. The results obtained from adding 0.25 μg/mL ethidium bromide to the gel only are shown in Panel B. To quantitatively compare the sensitivity of each method, gel photographs from two separate experiments were analyzed with the Phoretix 1-D gel analysis software package (Phoretix International, Newcastle-Upon-Tyne, England, UK). Figure 2 shows the relative fluorescence intensity of each amount of DNA on gels stained with the two protocols. Values are expressed as a percentage of that obtained for the 500-ng DNA band on each gel. With both methods, DNA could be detected at levels as low as 16 ng, although the background fluorescence was slightly greater on gels stained with the standard protocol (Figure 1A). The apparent small difference in relative intensity obtained with the two procedures (Figure 2) was not statistically significant. We have routinely used the modified procedure described here to stain DNA fragments over a size range of 170 bp–50 kb and have found its sensitivity to be comparable to, or slightly better than, the normal procedure over the whole size range. Lower concentrations of ethidium bromide (0.1 μg/mL) were tested but found to be less effective at staining DNA fragments smaller than 2 kb (not shown). This is presumably because there was insufficient ethidium bromide present to fully saturate the available sites in the DNA. Our data show that 0.25 μg/mL of ethidium bromide added only to the agarose gel is sufficient to detect DNA with comparable sensitivity to the standard method. Adding ethidium bromide to the running buffer is therefore