I also have question involved with transfection using PEI. In my experiment, I try to transfect my DNA to SY5Y and 293T cells using PEI. In 12-well plate, I used 1.6ug DNA in 100ul OptiMEM mix with 4.8ug PEI in 100ul OptiMEM, then mixed them, stand at RT, 20min, then, drop the mixture to the 70-80% confluence cells (containing 1mL of DMEM, 10% FBS, no antibiotics). Then, I changed media after 7h of transfection. After 48h, I observed good signal of GFP signal. However, I found that the confluence of my transfected cell still not increase (still 70-80%), whereas the Control (with PEI but no plasmid DNA) was completely over 100% confluence. I worry that this will affect to my result. Is there any one have experience on this? Is that because of too much DNA using for transfection? Thank you.