transfection of 293T w/ SuperFect - help, need info (Jun/14/2005 )

Hi all,

I am trying to do a transeint transfection of 293T cells with DNA constructs to then look for the expression of the prootein with Western blots. The cells are great growers, but when I plated 1x10^6 cells in a 10cm dish, the confluency the next morning was barely 50%. I went ahead with the transfection, but am concerned that the confluency will lower efficiency. Does anyone have experience transfecting these cells, and if so, how many cells do they plate the night before (I need to grow 48 hours after transfection)?

thanks

I used 293T in this way: 48 after transfection i plated 1x10e6 cells in a 10cm dish with 10 ml of MEM medium without antibiotics.

Thanks so much!!!

I just have another question. Did you mean you plated 1x10^6 cells after transfection or the night before? If it was after, how many cells did you plate for transfection? Did you take the antibiotics throughout the whole experiment? Is it to make the conditions less harsh?

You should plate enough cells (about 85%-90% confluency) into the dishes
20-24h before the transfection, so that the cells grows (about 95% confluency) when you transfect. By the way, you'd better culture 293T cells with better FBS.

Thanks so much!!!

I just have another question.  Did you mean you plated 1x10^6 cells after transfection or the night before?  If it was after, how many cells did you plate for transfection?  Did you take the antibiotics throughout the whole experiment?  Is it to make the conditions less harsh?

Thanks guys!!! I will try the suggestions