Electrophoresis zyxwvutsrqponm 1996, zyxwvutsrqponm 17, 1537-1541 zyxwvutsrqpon Zinc-imidarole negative staining of nucleic acids 1537 z K Eugenio Hardy Elder Pup0 Racmar Casalvilla Angela E. Sosa Luis E. Trujillo Eduardo Ldpez Lila Castellanos-Serra Center for Genetic Engineering and Biotechnology, Havana City, Cuba Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide or agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual ele resis reagents, then incubated in 10 mMzinc sulfate for 10 min, and subse- quently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipi- tate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids rose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, fol- lowed by immersion in 0.2 M imidazole for zyxw HGF 5 min to form a deep white-stained background. On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions where nucleic acids are lo resulting in a negative image of colorless and transparent nucleic a against a white background. The sensitivity of this stain ranges fro 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/ band for dif- ferent 22-base oligonucleotides, from 62 to 125 ng/band for large (fro 20 to 2 kbp) DNA, and is 1 pg/band for human peripheral-blood monocyte RN After chelation of zinc with EDTA, the nucleic acids can be quantit recovered from the gel. The principal advantage of this technique o dium bromide staining is evident for preparative purposes. Using zin zole in the detection of purified pBACIB.l (2.8 kbp) plasmid DNA an HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtaine conventional ethidium bromide staining plus UV-irradiation at 3 12 nm) to 10 000. 1 Introduction In molecular biology, the most popular detection method for nucleic acids is staining with ethidium bro- mide, followed by gel observation under ultraviolet (UV) light [l]. Although this method is rapid and simple to perform, as well as highly specific for nucleic acids and sensitive enough for most purposes, it has two important disadvantages: (i) ethidium bromide has carcinogenic effects that complicate handling and disposal of both this chemical and the stained gels, and (ii) the UV light generally causes deleterious nicking, dimerization, and bleaching of nucleic acids [2]. Even when using a UV light source with an output of 300 nm, which has been shownto provide a relatively low amountof photo- damage and photobleaching, the biological activity of plasmidic DNA has provedto be jeopardized [3]. In order to minimize environmental pollution and to pre- serve chemical integrity and high biological activity of nucleicacids,we have developed an imidazole-zinc staining [4] for DNA detection after agarose gel electro- phoresis. This is a two-step procedure by which the agarose gel is soaked in an imidazole solution followed by zinc salt staining. DNA is detected as a pattern of white-stained (positive), and transparent, colorless (neg- ative) bands contrasting against a deep white-stained gel background [4]. However, while majornegative DNA Correspondence: Dr. Eugenio Hardy, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, La Habana-6, Cuba (Fax: +537- 218070; +537-336008; E-mail: zyxwvutsrq quifis@ingen.cigb.edu.cu) Keywords: Zinc staining / Agarose gel electrophoresis / Detection of nucleic acids / Zinc-imidazole detection / Negative staining bands are well visualized, the image contrast betw positively stained bandsand the background staining was extremely weak, lowering the sensitivity of detection of thecomplete DNA pattern. Therefore, we recently combined the zinc and imidazole salts in a different manner, permitting the positive staining of DNA ban on agarose gels zyxw LKJI [ 5 ] . This zinc-imidazole positive staining method is relatively simple (4 steps) and applicable t wide variety of nucleic acids in a wide range of m ular weights (from 1 to 12 kbp). Furthermore, it causes no irreversible chemical modification of nucleic acids, allowing quantitative recovery from gel slices for su quent characterization or use. However, the question remained whether these biom cules could be visualized as homogeneous, negatively stained patterns, as previously observed with proteins [4, 6, 71. We describe herein a modification of the zi imidazole positive method that makes the homogeneou negative staining of small (< 1 kbp) DNA on polyacryl- amide gels possible. Additionally, negative staining successfully extended to the detection of nucleic a on agarose gels. This procedure has been validated by comparing the nucleic acid patterns with those obta using standard ethidium bromide or methylene blue stains. The principal advantage of this technique over ethidium bromide staining is demonstrated for prep tive purposes. Using zinc-imidazole in the detection of purified pPACIBl (2.8 kbp) plasmid DNA and anti- HBsAg single chain Fv antibody fragment (0.7 kbp) DNA by elution from gel slices followed by ligation and tra formation of competent E. coli XL-1 Bluecells, the numberof transformants notablyincreased obtained z F 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1996 0173-0835/96/1010-1537 $10.00+.25/0