Double - Triple stable transfection - (Jul/12/2007 )

hi all,
I have 3 different plasmid (same vector but different inserts) and I want to check a possible additive effect targeting in different places my gene. I'm gonna do, or try to do, a double and triple transfection with Fugene but I have few questions:

- do I have to transfect them in different days?
- normally I use 6 ugs DNA, shall I use 6 ugs each?
- should I expect that the double-triple transfection will be more toxic than the single one?
- can I use like control my single transfected vector only even if those cells won't be stressed in the same way?

thanks to everyone who will answer

Seb

-Sebastiano-

I'm not sure on your actual questions....however, in your post title, you say stable transfection. You won't be able to select for each vector stably unless each has a different resistance marker.

-miRNA man-

Hi Seb, when I did double transfections I used the same total amout of DNA as I would for a single transfection so therefore in your case I would have used 3ug of each (or in a triple 2ug of each). This should not be any more toxic as you have the same total amount of DNA but you can check with a toxicity assay. Hope this helps!

-auldmok-

QUOTE (Sebastiano @ Jul 12 2007, 02:39 AM)

Seb

We frequently transfect cells with 4 plasmids at the same time.

We keep the total DNA the same, so if you would use 6 ug for 1 plasmid, for 2 plasmids use 3ug each

I havnt had toxicity problems with transfections atleast compared to a single or triple transfections. I had some problems initially while optimising transfections.

Good Luck !!!

-scolix-

it's possible to check which cells have 2 or 3 inserts just with a PCR, I designed primers specific: FW binds my insert and RW further down in the vector, so if I get a band my vector is there; I won't run them in multiplex but it's the easiest way to do it.

thanks for the answers, I'll do as auldmok and clolix said!

-Sebastiano-