Hi everyone,
I am currently isolating plasmid encoding a viral envelope protein that will be transfected into a mammalian cell line in order to make virus. After I ran a gel to monitor my plasmid isolation, I see that I have 2 bands above, which I am thinking are most likely the nicked form and a relaxed circular form (I don't think it's genomic DNA because the digested bands look very clean). I definitely have ideas on fixing the purification (culture for less time and be more gentle with isolation), but I was wondering, in general, does the conformation matter in terms of transfection efficiency if I am going to be using something like Lipofectamine? Also, would a cell be able to fix the nicks in plasmids? Thanks in advance! I've also attached a picture of my gel, don't mind the dots, our staining tank is very dirty
What I know is that only the high-quality plasmid could get high transfection efficiency.
In general, supercoiled plasmid DNA gives higher efficiency, with more transfected cell per microgram. However, if you want integration and sustained expression, linear plasmid does better after selection, as sells that have the selectable marker are more likely not to have a break in the expression cassette. Supercoiled plasmid has to become linear to integrate, and can break anywhere outside the resistance cassette, so many do not express the gene of interest. What is best depends on transient or permanent expression, whichever is your plan.
better aim for max percentage of supercoiled plasmid of course, but realistically you dont need to deliver a lot of plasmid into the cells- as a ton of RNA and protein will be produced from it (delivery of eg synthetic si/miRNA is another story, there is no "amplification" in this case). Lipofectamine 2000 and 3000 are fantastic reagents, working in a broad range of cells, and after a brief optimization of transfection conditions- you will get top results, guaranteed
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