Transient Transfection of 293T - (May/25/2006 )

I did transient transfection with 293T cells to overexpress my protein. I used lipofectin method and used MSCV-IRES-GFP vector, which is easy to estimate transfection efficiency by looking at GFP positive cells.

Interestingly, the cells transfected with empty vector showed almost 100% GFP positive under fluroscence microscope one day after transfection. However, all the other 3 transfectants, with my constructs in, showed few GFP cells, which I can't distinct with the background.

Have u checked your DNA preps, incase they were labelled wrong.

Do you think the insertion of a cDNA upstream the IRES could alter the transcription? I promise my DNA is correct. But someone said my DNA quality could be bad. How can I check it?

I apologize, I misunderstood u.

Its possible that GFP might not be expressed properly, the reason could be IRES sequence. They can behave this way.
Also the quality of DNA is also important for transfections.

Measure the DNA conc. using spectrophotometer and it also gives u 260/280 value.
I dont know the exact values for purity, but if its more than 1.7 or 1.8 , then its clean.

Also run in on gel to check for contamination with RNA. this could also limit transfection.

Good Luck !!!