Hi ,
i tried many transfection reagent and Viafect worked best for me when i used in HELA cell (knockdown for some protein) as observed by reporter plasmid (5.4 kb). When i do my experiments i have to add 3 plasmids : reporter plasmid + 2 filler plasmids (6kb) -i get efficiency reduced to 40%. However, when i use reporter plasmid + 2 effector plasmids (8 kb) i hardly get any GFP positive cells efficiency hardly 1%. does it has anything to do with size of plasmid. How should i optimize transfection as i guess 8kb is not very large size.
Any help will be highly appreciated.
Thanks
You may need to decrease the total amount of plasmid DNA ( a max of 10ug)/plate.
Okay,
I remember I used to transfect 4 different plasmids in a total of 10ug into HEK cells in a petri dish. But I was using a manual transfection methods (calcium phosphate).
Good luck.
Hi Atoshi,
I have regularly been co-transfecting HEK293T cells with three adenoviral plasmids, two of them >9kb and one of ~6 kb. I have in the past used BioT transfection reagent, but as of now using Jetprime transfection reagent which is relatively much better and the overall transfection efficiency is also very good. You may try Jetprime and I hope it would work in your system as well. All the best!
I normally use Xtremegene9 transfection reagent for my experiments and it seems to work well. One thing we have noticed in the lab is that if the cell density is low the plasmids can have a cytotoxic effect with respect to Hela cells. I am not sure about your cell density to plasmids ratio, may be it could help if you optimize this part .
Lipofectamine 3000 is the best option u might need as a transfection reagent.
thank you all for the replies. I re-purified my plasmids as i believe the plasmid isolation kits are not transfection efficient especially for large plasmids and to my surprise i got a very good transfection.
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