Appendix D - Disposal of Ethidium Bromide and SYBR Green Solutions

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A P P E N D I X

D

Disposal of Ethidium Bromide and SYBR Green Solutions

Ethidium bromide (EtBr), SYBR Green, and similar dyes that are used to stain nucleic acids in gels are very powerful mutagens (MacGregor and Johnson, 1977). As intercalating agents and nucleic acid-binding dyes, they should be treated as toxic waste; the proper handling of these materials includes prevention of environmental contamination after use. Only after proper treatment can EtBr and SYBR Green-tainted wastes be disposed of with minimal apprehension. The salient issues surrounding the treatment of EtBr waste have been described at length (Lunn and Sansone, 1987, 1990; Bensaude, 1988). The widespread practice of adding bleach to ethidium waste is not recommended, because such treatment, although reducing the mutagenicity of EtBr, converts the dye into another mutagenic compound (Quillardet and Hofnung, 1988). Instead, one of the following protocols should be adopted as standard laboratory policy and applied to the handling of SYBR Green as well. The following protocols suggest the handling of working concentrations of EtBr (0.5–1.0 µg/ml) and SYBR Green (1–4×); at higher concentrations, these dyes can be diluted down and processed as described in Protocol 1, Protocol 2, or Protocol 3. Protocol 1

The Extractor (Scheicher & Schuell; Cat. No. 448031) is a one-step filtration method for the removal of EtBr and SYBR Green from gel-staining

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solutions (Fig. D.1). The device is capable of filtering 10 liters of electrophoresis buffer containing EtBr, with greater than 99% removal (Fig. D.2). The filter, an activated carbon matrix, is disposed of according to departmental guidelines, whereas the filtrate can be safely discarded down the drain. This is an extremely cost-effective method for dealing with this common waste product in the molecular biology laboratory and is used extensively in this laboratory.

Ancillary Protocol

Quantitative assay for residual EtBr in filtrate (Menozzi et al., 1990): 1. Prepare a solution of 100 µg/ml salmon sperm DNA in running buffer or staining buffer. Prepare dilutions of EtBr in the range of 0 to 500 ng/ml to prepare a standard curve. 2. Add salmon sperm DNA to 1-ml aliquots from each volume of filtrate to a final concentration of 100 µg/ml. 3. Read standards and unknowns in a fluorometer (excitation 526 nm, emission 586 nm). Plot the standard curve and read the EtBr

F I G U R E

D . 1

Extractor filtration device for the removal of ethidium bromide and SYBR Green from electrophoresis and staining buffers. Photograph courtesy of Scheicher & Schuell Bioscience.

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Disposal of Ethidium Bromide and SYBR Green Solutions

F I G U R E

D . 2

Ten successive liters of buffer containing 500 ng/ml of EtBr were filtered through the Extractor device. Residual EtBr in the filtrate was measured by fluorescence spectroscopy. Data courtesy of Schleicher & Schuell Bioscience.

concentration of the unknown from the standard curve. This assay is usually linear (r = 0.999) for EtBr concentrations as low as 4 ng/ml.

Protocol 2

Solutions containing working concentrations of EtBr, SYBR Green, and related dyes can be decontaminated by adding about 3 g of Amberlite1 XAD-16 (Sigma Cat. No. XAD-16) for each 100 ml of solution (Joshua, 1986; Lunn and Sansone, 1987). This resin is a nonionic, polymeric absorbent. The solution may be shaken intermittently for 12 to 16 hr at room temperature, after which it is filtered with Whatman No. 1 filter paper or the equivalent. The filter and Amberlite should be treated as toxic waste and disposed of according to in-house laboratory policy. The filtrate can then be discarded.

Protocol 3

Add 100 mg of activated charcoal (Sigma Cat. No. C-2889) to each 100 ml of EtBr or SYBR Green at the working concentration (Bensaude, 1

Amberlite resins are manufactured by Rohm and Hass, Inc. (Philadelphia, PA).

References

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1988). The resultant mixture may be shaken intermittently for 1 hr at room temperature, after which it is filtered with Whatman No. 1 filter paper or the equivalent. The filter and charcoal should be treated as toxic waste and disposed of according to in-house laboratory policy. The filtrate can then be discarded.

References Bensaude, O. (1988). Ethidium bromide and safety—readers suggest alternative solutions. [Letter]. Trends Genet. 4, 89. Joshua, H. (1986). Quantitative absorption of ethidium bromide solutions from aqueous solutions by macroreticular resins. BioTechniques 4, 207. Lunn, G., and Sansone, E. B. (1987). Ethidium bromide: Destruction and decontamination of solutions. Anal. Biochem. 162, 453. Lunn, G., and Sansone, E, B. (1990). Degradation of ethidium bromide in alcohols. BioTechniques 8, 372. MacGregor, J. T., and Johnson, I. J. (1977). In vitro metabolic activation of ethidium bromide and other phenanthridium compounds: mutagenic activity in Salmonella typhimurium. Mutat. Res. 48, 103. Menozzi, F. D., Michel, A., Pora, H., and Miller, A. O. A. (1990). Absorption method for rapid decontamination of solutions of ethidium bromide and propidium iodide. Chromatographia 29, 167. Quillardet, P., and Hofnung, M. (1988). Ethidium bromide and safety—readers suggest alternative solutions. Trends Genet. 4, 89.
Appendix D - Disposal of Ethidium Bromide and SYBR Green Solutions

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