Gencarrier-1 vs. CaPO4 transfections - (Mar/09/2009 )
I purchased Gancarrier-1 and used it to transfect HeLa cells with pGL-3 vectors for a few time. No luminescence has ever been detected at all. Then I switched to CaPO4 method. For the first time I got some luminiscence, although the conditions might need to be further optimized to improve the efficience. Now my question is: Is CaPO4 method only suitable for certain cell lines and certain studies? My boss doubt that Ca would trigger the signalling of some pathways in these cells.
On the other hand, I still don't know why my transfections with Gencarrier-1 always failed. Can somebody tell me whether there is some trick for your success of utilizing this reagent? Thank you so much!!!
Never used your reagent, so I haven't an optimal protocol. Try the DNA reagent of synvolux (you can get a free sample on their website).
I've had good luck transfecting with CaPO4 with primary neuronal cells... much better efficiency than regular lipofection reagents. It is possible that CaPO4 would work best with your cell line.
But you might want to try some other reagents that have been shown to work well with your line.
with most liposome based transfection reagents it is important to titrate both the amount of DNA being added and the amount of transfection reagent. HeLa are not hard at all to tranfect with many reagents. I use Fugene and Lipofectamine commonly and have never had a problem using standard conditions (1ug plasmid and 2 ul/ml fugene per well of 6 well plate).
Many of the protocols for these reagents are based on area of plate or dish rather than the amount of DNA being transfected, so check your conditions and see what you did.
I never used Gencarrier-1, so no comment on this. I could give you some personal opinion on CaPO4 transfection. The PH of buffer in CaPO4 transfection is very important. Base on varied condition in different labs (PH of ddH2O, PH calculator), PH suggested in a protocol should not be always followed. In our lab, we always prepare many buffers with a series PH (7.0,7.2,7.4,7.6) and try which PH is most suitable for a high transfection efficiency. If possible, you should consider this. May it help you. Good luck.