I have an E. coli cell line I use to express a protein using a two plasmid system. One confers AmpR and one KanR.
For mutagenesis I would like to separate the plasmids to increase efficiency of the mutagenic PCR reaction. I was able to cure the cells of the KanR plasmid because it is low copy whereas the AmpR is high copy so I could easily grow the cells in ampicillin only and I ended up with only the AmpR plasmid. However I was unable to do the same to isolate the KanR plasmid.
My next idea was to run the plasmids through a gel and do an extraction. Since I didn't do a digest it was very hard to differentiate the bands and I ended up getting a negligible yield. I believe the plasmids were simply smeared through the gel due to tertiary structures.
My final hope is to perform a digest on the plasmids using a restriction enzyme that cuts each plasmid once. My concern is that I will not be able to get the stoichiometry correct due to the plasmids both being simulatenously present in vastly different concentrations.
Does anyone have any tips on how else I could isolate the plasmids? Or maybe does anyone have experience running a digest on plasmids with unknown concentrations? I really need help doing this. The mutagenesis project has been going on for months and this isn't even a molecular biology lab... so it's detracting time from main research goals!