I need to optimize a transfection protocol to transiently express a plasmid encoding a chimera of eyfp attached to the c term of a Golgi apparatus signaling molecule) in hela cells and hepg2 cells and get as high expression as I can get.
I need enough protein for about 100 wb's. I've seeded hela cells to 12 150mm dishes. And the same with the hepg2. I've prepped mg's of plasmid.
I'm using invitrogens original lipofectamine reagent; not the 2000, or ltx or any of that stuff.
Any suggestions on a protocol here for hela and hepg2 plasmid dna and lipofectamine mediated transfection on such a large plate size.
I figure just scaling up should work but there's some pitfalls here. Look at this table for example when you go from 60 to 100 mm you about double the area, so you also about double the culture volume. However when you go from 100 to 150mm you triple the area, but only increase the culture volume two fold about. This is making my scale-up calculations for transfection look really off: