what is the best way to set up the transfection? - (Oct/31/2005 )
I wondered that how you guys set up your transfection. do you set up like fix cell number, then change the ratio of DNA and lipofectamine. and also change the cell number and fix the DNA and lipfectamine ratio. I am going to do stable transfection. so I wondered that should I set up 24 well with diffenrent ratio of cell number, DNA and lipo ratio,
To start with keep the cell number constant. If you vary the cell number..especially when it is low then u might have toxicity due to the transfection agent...
Ideal thing is to keep the cell number constant and vary the DNA and lipofectamine. Also try ur transfections in antibiotic free media...I doont know why but it seems to work..
Another thing you can do is to compare the influence of Serum during the transfection. Some cells will not transfect efficient in the presence of serum. Once again, keep all other parameters constant when comparing a with and a without serum transfection.
If you experience toxic effects: chech how soon you can remove the lipofectamine and DNA. Invitrogen advises 4-6 hours, but less than 3 has worked for me. Again, do this on the same number of cells, the same amount of DNA,...