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 CaP 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!
More information about the Methods