transfection of Hek 293A cells - (Oct/12/2005 )
I'm trying to transfect the Hek-293A cell with plasmids and I found that different plasmids have dfferent efficiency. The one which I hope for better efficiency truns out to be around only 30%.I used Lipofectamine, and transfect half of the optimum amount of DNA as asid in instruction. Could anybody give some suggestions on improving it at least to 60-70%?
are the plasmid preps endo-free?
Yes, all are
So that was the easy answer, I haven't worked with HEK cells, but when I was trying to improve transfection efficiency I just did multiple transfections with different reagents I borrowed from neighbors or got as a free-trial. It may not be relevant to your cell line, but Superfect worked the best for me and I got at least 70% with it... Use something like GFP to make the comparison of efficiency easier... Another possibility is using the reagent you have now and playing wiht the DNA concentrations or the ratio of reagent to DNA etc. I know this is alot of work, but I can't think of any simple way.... Maybe someone else can suggest something....
I hope this helps, good luck!!
i've noticed using 293 cells that the best ratio DNA lipofectamine was 1µg DNA for 3µl lipofectamine. I get better results when starting at 50% confluency.
I let the mixture 5h then add 2Xserum medium (DMEM medium (WHITHOUT any antibiotic and without any complements)
Then 24h after start i add fresh complete medium
I have used lipofectamine 2000 with these cells and got ~99% transfection effiency measured by FACS. I also use the 1ug DNA to 3ul lipo2000 ratio. I let the mix sit on the cells for 6 hours, remove, then add media + serum (pen-strep only). I measure EGFP expression 48 hours later. Good luck.