Stable transfection of large plasmid into breast cancer cells - (Oct/18/2006 )

Right, the little blighters are annoying me! I've managed to successfully get the cells transfected with a GFP - only labelled plasmid, they are nice and green. However, the cells that have been transfected with GFP+gene of interest, which are large plasmids at about 5.5kb, are not glowing green. The antibiotic selection isn't working either.

Are there any suggestions on how you can stably transfect a large plasmid? Do i need to leave the transfection mix on these cells for longer (i'm currently leaving it on for 5 hours to minimize cell death)?

have you done a kill curve for your antibiotic.

5.5 kb is average size for routine plasmids. Size upto 15 kb can be transfected reasonably well with lipid-based reagents, such as Lipofectamine 2000. Total cellular uptake with labled plasmid means nothing if transfection outcome is the main interest of yours. I would not move further if I dont see green EGFP positive cells. What cell type are you working with?