I am co-transfecting 3 plasmids, respecting the maximum amount of DNA per volume and using a established DNA:cationic reagent ratio in 293T cells.
One of my plasmids has a fluorescent marker and I can access the transfection efficiency for it using flow cytometry, which is >85%. Is the safe to assume that the other two plasmids are being co-transfected with the same efficiency?
Hi Danuta, I can confirm Ondrej's answer. We have a lot of R&D data where we cotransfected a surface molecule (MHC.H2KK receport) and GFP. If we use the same amount of molecules, we got exactly the same number of double positive cells. Again, that is my observation with electroporation or Nucleofection.
Thank you for your answers! I've observed 65% triple-positive 293T cells - but one of the plasmids is DOX inducible, so it may not directly reflect the co-transfection efficiency.
I think the other two plasmid will be co-transfected as well with the same efficiency (if the they have nearly the same size) and the best example for that is lenti-virus production .. you co-transfect 3 plasmids with same DNA amount or molar ratio (2 packaging and one transfer plasmid which may have a reporter ) and the virus is packaged again from these plasmids. so I think it should be fine. all the best!
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