I know the general process for transfecting a plasmid, however, how is it with two plasmids? Do I have to perform PCR first to create one combined plasmid or would I just use both plasmids when transfecting? I was looking into tagged plasmids that have GFP for example, to make it easier to detect whether transfection worked or not. I will use 3T3 cells, which are generally harder to use for these experiments from what I read. I'd appreciate any advise or literature I can look into.
Hi Anna,
You should have no problem transfecting both plasmids at high efficiency into the same cell. Most reagents like lipofectamine, similar products, and even electroporation, often have a co-transfection rate of over 90%. Having said that, the only way to assure that a cell has both of your plasmids is to have a way to detect products from each plasmid in a cell, i.e. GFP-tag or another, that you mentioned. However, this may not be necessary depending on your experimental assessments. I have done these kinds of experiments a lot and would be happy to follow up if you had any questions?
Best,
Daniel
Hi,
Another possibility would be expressing the complex recombinantly and then tranfecting the cells with purified protein complex directly.
Best
I have also had good luck using both plasmids at the same time. .
Using Lipofectamine.
You can run a PCR on a cell lysate to check for your inserts of interest and if they are over-expressing.
Best Wishes,
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