Isolate plasmid from eukaryotic cells after transfection - (Aug/26/2009 )
I inject/transfect plasmids into eukaryotic cells. My problem is, that I want to get them out again after the procedure and need them to be really clean. I tried to use the standard plasmidprep procedure for bacteria, but I also get a lot of genomic DNA (as determined by qPCR) which I must not have for my downstream applications.
Has anybody ever isolated pure plasmids from eukarytic cells and if so, how did you do it?
Thanks for your help in advance!
Why do you need to get them out again? If you have the plasmid in bacteria you can make more very easily and use that.
The problem you will find is that the plasmids that persist in the cell have linearised and integrated into the host cell DNA - it will be hard to get them out.
Of course it is easier to isolate the plasmid from bacteria.
But for my experiment, it is crucial to have the plasmid in the eukaryotic cell for a defined time. I am interested in DNA modifications and want to see what the cellular machinery is changing while the plasmid is inside the cells. For this, I have to analyse the plasmid afterward it has been inside the cells and this analyses only makes sense if the plasmid is really pure, as genomic DNA will contaminate my assay system.
I am still hoping that someone can help me out on this one!