Julie Auger asks: >Has anyone tried sorting chromosomes using Acridine Orange and 7-AAD? We >want to sort Indian muntjac chromosomes (only 3 or 5 chromosomes per >cell). Any advice would be appreciated. Why AO and 7-AAD? If the idea is to get a 2-color bivariate histogram, AO probably wouldn't be the best choice because it has high background fluorescence, is metachromatic, and - if I remember correctly - doesn't have much base pair specificity. I've tried 7-AAD on chromosomes, and the staining is very weak, and not, at least in my hands, particularly base-pair specific. Both Tom Frey at B-D and I got some bivariate staining of human chromosomes with combinations of 488-excited and red-excited Molecular Probes dyes (TOTO-1 and TOTO-3 in my case, thiazole orange and thiazole blue in Tom's), and that might be worth a shot. These dyes aren't DNA-specific, but chromosomes don't have appreciable RNA. But maybe the whole bivariate thing is overkill; I seem to recall that Muntjac chromosomes can be well separated using a single stain, such as PI. It might be that a single Molecular Probes dye, such as Pico Green, would be equally useful. -Howard
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