I want to linearize a plasmid and just realized that there is no blunt end restriction site available in a non-relevant plasmid area.
Do you think it is relevant if I cut the plasmid blunt or sticky? My first guess is that the mechanism of integration probably differs but both sticky and blunt end plasmids should be integrated. Is there a study out there that addressed this question?
Hi Friedrich
in our lab we have been performing stable transfection now for 3 years without linearization and it is working fine. so, may be try it just like that. if you would like you can perform the transfection with both the linearized (blunt or sticky does not matter) and coiled plasmid and check for the difference of response.
Works well for coiled plasmid, linearised sticky and blunt: things are a bit different according to sequence but not much. If you are worried then do the lot (if you can be bothered) and see which works (provided your plasmid DNA isn't the limiting factor).
For anyone that wants a bit more solid info on this, I found this article where linearised and non-linearised plasmids were used and cleaving can improve the efficiency of generating stable transfected cells and more importantly, perhaps, integrity of whatever the plasmid will express. The site of cleavage seems to play an important role, too. Cutting pEGFP-N1 with BsaI improved things over the uncut version, while cutting with SspI reduced transfection efficiency, %of positive clones etc dramatically.
Article Optimizing the generation of stable neuronal cell lines via ...
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