We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by pulsed field minigel electrophoresis. The best experimental conditions were as follows: separating large DNA molecules in minigels of 0.4cm thickness, further incubating them with 40mM ZnSO4solution, and finally incubating them with 0.1 and 2M imidazole solutions successively. The lowest yeast cells/miniplug useful in DNA band detection was 3×107cells, as occurred with ethidium bromide-stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves the chemical integrity of the DNA molecules. © 2010 Elsevier Inc.
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