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 
-- 
    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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