Co-transfection - (May/07/2010 )

Dear all,

I want to transfect (transient) my cells with a dominant negative mutant. This mutant has a nice tag to detect. Actually I would like to co-transfect the cells with a plasmid carrying my protein of interest to see the effect of the mutant. Unfortunately this plasmid has no tag. Now my question is it necessary that this plasmid has a tag or can I run the experiment? I tranfect HEK cells.
Thanks for all comments
Purzel

Purzel on May 7 2010, 09:28 AM said:

Theoretically, if the cell has taken up one plasmid, 90% of the time, it would have taken up the other. So, to answer your question, it would be entirely possible to use one plasmid as a determination of successful co-transfection. Do keep in mind, that there is a small chance that the cell is only expressing one plasmid.
If the experiment is repeated enough times, the chance will become statistically insignificant.

Hope that helps!

Labrat612

Hi,

Just out of curiosity, what is the theory behind this? I mean, if that was a stochastic process, then a transfected cell has either both or only one plasmid. That would mean that almost 70% of transfected cells are not co-transfected... From my experience, co-transfection does not result in 90% efficiency, but rather 75% or even worse. I'm wondering if anyone has ever tried to quantify this.

Cheers,

Minna