293 transfection

[Richard J. Dudley]

I am transfecting 293 cells (human epithelial kidney) with Lipofectaime 
fron Gibco-BRL.  The first time worked splendidly, but subsequent 
transfections (we're using transients) have yielded mediocre results.  
There are several superstitions I have heard, one of which is something 
to the effect of, if the primary culture exceeds approx. 70% 
confluency, subsequent generations of cells will be affected negatively 
in terms of transfection.  The solution is to thaw an aliquot of early 
generations and start again.  I have tried several other solutions--I 
was wondering if anyone could provide some insight into this one.  My 
primary did reach approx. 90% confluency on one ocassion.  I have many 
early generations frozen away, so if this is true, there's not much of 
a problem.  Thanks to anyone with info, and I'll post what I find out 
later.

rich

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Richard J. Dudley
Neurobiology Research Center
University of Alabama School of Medicine