Zinc-imidazole negative staining of chromosomal-sized DNA molecules in agarose minigels

Abstract

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 ZnSO4 solution, 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.