Hi there,

Yeast Plasmids are usually nuclear so there is no way to spot them using DAPI because of the chromosomal staining signal. What is usually done using plasmid containing lacO sequence is to express LacI-GFP fusion and to visualize the plasmid using GFP fluorescence. I guess your DAPI staining spots outside the nucleus are mitochondrial DNA which you should be able to colocalize with specidfic mltochondrial staining.

@Dominique Liger Thank you for your answer, however, just out of curiosity, what could be the structures lying outside of nucleus stained with DAPI in the transformed yeast cells (containing episomal plasmids) as they can hardly be mitochondria because I could not observe them in the wild type yeast (devoid of any plasmid) and since as you said its not possible for them to be plasmids either.

Well, DAPI is actually staining DNA (but not the whole nucleus actually, for instance nucleolus is much less stained than the rest) and the intensity of the staining is depending on DNA compaction state. In your case it might be that in your transformed strain centromeric regions are more compacted than in the wt strain giving some dots as a result. How sure about these dots being not nuclear (did you try simultaneous stains on the same sample for DAPI and mitochondria specific staining for instance) ? At what stage of the cell cycle do you observe these dots? There are definitely different possibilities to explore...

@Dominique Liger, Thank you for your insights. For one, when I said stained structures outside of nucleus, I meant they are distributed well outside of what the nucleus would normally occupy in the cell. They were well dispersed inside the cell. At the moment, I do not have mitotracker with me, hence, I am unable to confirm whether these are just mitochondria or cellular debris or as you said nuclear material..