Transfection-help! - (Aug/05/2007 )
I have a question. Does any one know that if there is anything inside a maxi-prep DNA kit that would presult in the DNA produced having a low expression in co-transfection. (The low expression is actuallly for another plamid which I didn't mak, when co traansfected with the plasmid i made and maxi-preped). The kit I used, is endotoxin free and is from invitrogen- pureink). It would be great if someone can help, as it would be great that the reduced expression (from just looking down a fouresent microscope) isnt random but means that some protein interaction is going on!
i dont think that maxiprep kit got anything to affect co transfection , i did it and tried triplet transfection and was ok, the main thing is to optimize ur plasmid amount in co transfection assay , try different wells with differenent amounts for each plasmid* note not to exceed the upper limit for plasmid concentration * and look for the best concentration for ur expierement.
hope this help
The thing is I used the same concentration of a EGFP tagged plasmid in each of the 2 co-transfections cocerned, the only thing changed was that I added a mutated version of another plasmid in my second transfection (not the GFP tagged one), and I got a lower expression of the GFP tagged plasmid. So I don't think that is a concentration problem? Help? Thanx
transfection protocols have to be optimized for each cell type and transfected gene; some neuronal cell lines and primary cells are difficult to transfect; use no-serum-containing medium such as OPTIMEM; some providers offer enhancer "Plus reagents" and so on to improve chemical transfection
Low expression, could be due to the promoter, transcriptional elements and cell type. And ofcourse, the transfection itself.
from the prep itself, well except solvents (ethanol at OD230) i don't really think preparation is the problem. For the column preps, generally proteins contaminations is a negligible level.