Multiple plasmid FuGENE 6 transfections
Ian A. York
iayork at panix.com
Fri Sep 21 13:46:54 EST 2001
(Please, when you both email and post a comment, indicate that you've done
so. I've already answered this in email, and I had assumed you had made a
private comment; now I have to repeat myself.)
DAVID JACKSON said:
> 12 plasmid transfections are not absurd. I do these on a regular basis to
Another guy has also pointed to a system with 8-plasmid transfection, so I
guess I'm wrong.
> In this case immunofluorescence is not required as the production of virus
> particles and CPE is the proof of protein expression. Maybe CaPO4
> transfection is the answer.
I suspect that this is more feasible because you can get by with a very
low final transfection efficiency--even if one in a thousand cells is
transfected with everything the virus should then amplify itself. In our
case we saw drops from 90% transfection efficiency (single plasmid) to
less than 40% (three-plasmid) based on staining for multiple markers.
CaPO4 is certainly worth a try; it's widely used in adenovirus recue,
though that only involves a couple of plasmids, but I have the impression
that it brings in much larger glops of DNA than other systems.
If you have good-quality DNA, I doubt the details of purificaton are
critical. You might want to try QiaGen's (or similar) endotoxin-free
purification systems, which in some cell types (but not all) significantly
improve transfection efficiency.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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