about stable transfection - (Jan/15/2007 )

I have several questions to consult：
1. Is the 6-well plate big enough for the initial transfection? About how many cells will be integrated with the inserted gene?
2. How can the quantity of plasmids infect the efficiency of both transfection and integration. It seems in my experiment that too much plasmids leads to low transfection efficiency.
3. How long should I select the cells? One month?
4. How many times should I select the cells? Someone suggested me select the cells for 3 weeks and then, let them grow without G418 for another 3 weeks, and at last, select them with G418 for 3 weeks.
5. G418 does kill lots of cells everyday. I do not know whether to change the medium or not if there are too many dead cells floating in the medium. Will they do harm to my positive cells?

Thank you for your reply~

1: 6 well is ok.
Assuming you've 500 000 cells for transfection, roughly about 250 000 (in easy transfectable cells, let's say 100 000 or less in most tough cells) are transected.
Integration in the genome is rare event so who can tell?...

2: too much plasmid act like titering the transfection agent. If you have a too high ratio DNA:agent, then the DNA lipid complex will be bad enough for transfection.
If you maintain the ratio, then it may be that there will be too much agent of transfection, which is toxic too cells.

3: depend of selection.
Puromycin achieves in 1 week, but i maintain at least 2weeks.
Neomycin is tough and 1month is ok to be quite sure. Also i maintain for 2month. If i have an IRES Neo vector, i maintain all time the cells are in culture.

4 : see 3

5 : definitely change at least every 2days. The cells are partially lysing xhich delivers toxic molecules in medium.