question about sequential transfection several plasmids in 293T and COS7 cells - (Oct/23/2013 )

Hi, 

i tried to do transfection with 4 different plasmid in 293T cells. But i can't transfect the 4 plasmids together. I need to express protein A & B from 2 plasmid first. And i need to transfect GFP-LacI and last i need to transfect a LacO reporter plasmid (~20K). I've tried several ways to do this but i can't achieve high expression of proteins. and the transfection efficiency of LacO plasmid is very low (<5-10%). I'm using lipofectamine 2000 the transfection. Can anyone have some good advices on the procedures?  thanks !

-K 

How are you transfecting your cells? Are you doing a transient transfection, waiting two days and then transfecting again?

Do you linearize your plasmids when you transfect your cell with multiple plasmids. If not, I would at least linearize the LacO plasmid. That guy is huge and the cell may have some difficulty taking it up.

I would start by transfecting your lowest transfection efficiency plasmid, selecting for it and then proceeding with the others. How are you selecting for your plasmids? Does each plasmid harbor a different mammalian resistance or are you doing downstream assays to check the expression of each plasmid?