I am making stable cell lines by transfecting my cells with plasmids that will induce over-expression of my gene of interest. After selection and obtaining single colonies, I will be confirming the presence of the plasmid by doing RT-qPCR/Western blot for my gene of interest. But I can't use these methods with the cells transfected with the control plasmid (empty backbone).
Is it necessary to confirm the presence of the control plasmid? Or is it safe to assume that the colonies will contain the control plasmid since the cells transfected with the over-expression plasmid all show increased expression and my untransfected cells all died during selection?
I was thinking of doing a quick "dot blot" and looking for my vector via amp or g418 expression. But the PI said not to worry about it. They are doubling well with G418, and my over-expressing line is working well, with our protein well over endogenous. I would keep them under selective pressure by keeping antibiotic in the medium just to be safe.
I have even plated non transfected vector and added G418 to watch them be killed just to make sure it is still working.
A enzyme digest of the plasmid should yield more information too
Best Wishes!
It might be 'safe' to assume that the colonies contain the control plasmid, but it isn't scientific, so best to be sure. Luckily it isn't an overly complicated procedure.
You could always try isolating the plasmid DNA from the cells and then just sequencing it (either in-house or send a quantity of it away for sequencing) to confirm.
Hi
You should normally find a common target on both plasmids like AMP resistance or so which you could use for PCR based typing.
With this method you could check presence of plasmid for both conditions control and test samples
Sven
Thank you all! I think I will test for the expression of the resistance markers
- Badges
- Science topic