Transient transfection

Brooks Robey r.brooks.robey at mcmail.vanderbilt.edu
Thu Oct 19 20:19:51 EST 1995


Most transient gene transfer protocols depend, in part, on exponential 
cell growth . . . could your cells be too confluent or even quiescent? 
If so, we and others have had good luck using polycationic 
poly-L-ornithine to effect gene transfer in quiescent cell monolayers. 
Also, anecdotal experience in our lab is that the pH of the 
HEPES-buffered saline (HBS) is crucial to the success of Ca€P pptn in 
some cell lines, & the manner & order that the HBS, DNA, & CaCl2 are 
combined can determine the coarseness or fineness of the ppt itself & 
impact on the effieciency of transfection. Finally, luciferase is very 
heat-labile & protease susceptible, so make sure that you are handling 
your samples appropriately & are storing them properly or processing 
them promptly . . . protease inhibitors are ALWAYS a good idea with 
luciferase reporter gene expression!

Good luck.





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