NEGATIVE STAINING WITH ZINC-IMIDAZOLE OF GEL-ELECTROPHORESIS SEPARATED NUCLEIC-ACIDS

Zinc and imidazole salts were applied for the detection of nucleic aci ds on either polyacrylamide or agarose gels. After electrophoresis, po lyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfat e for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colo rless bands. Staining of nucleic acids on agarose gels can be performe d by incubation in 40 mM zinc sulfate for 10 min, followed by immersio n in 0.2 M imidazole for 5 min to form a deep white-stained background . On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc p recipitate is removed from the positions where nucleic acids are locat ed resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain r anges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 n g/band for large (from 20 to 2 kbp) DNA, and is 1 mu g/band for human peripheral-blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel, The princi pal advantage of this technique over ethidium bromide staining is evid ent for preparative purposes. Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain F v antibody fragment (0.7 kbp) DNA, followed by elution from gel slices , ligation and transformation of competent E. coli XL-1 Blue cells, th e number of transformants notably increased from 280 (obtained with co nventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10 000.