Hi all, I need to do co-transfection in RAW cells by pcDNA3 in which I have neomycin selection, I already have my gene of interest with flag tag and one with his tag in the same vector.
I have only one selection marker for Eukaryotic cell line, so is i am going to face difficulty in selection.
You know it will be difficult and you will never get homogeneous population.
I have never tried it but would strongly advise against it.
If at all doable, it would be concentration dependent and you should do sequential transfection and selection, I think. That is, first transfect the one plasmid and select with neomycin comparing to a non-transfected cells until you are sure you have stable genome integration. Then transfect the second plasmid, increase the neomycin concentration and compare to your line containing only the first construct. This is pure speculation, I have never tried it.
Rather go into the effort to exchange the resistance marker for a Puromycin (or Blasticidin) resistance.
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