FuGene 6 transfection problems - (Mar/06/2008 )
I want to know how true it is for the story that the transfection shoule be done without P/S. Did you test this rKay447 with P/S and found no good?
I used it with different cell lines. I think 1:3 ratio in 6 well plate was teh best for me with all cell lines I used. No media change and I still see expression upto 3 days (72 hours). yeah 50 to 70% confluency.
Complexing was from 30 min up to 2 hours and still working fine
USE beta gal reporter plasmi to optomize your treansfection
don't change media
Yes. When I first started I accidentally did a transfection with the full media and about 80% of the cells (I assume the ones that were transfected) died and were floating in the media.
Hey guys,
thanks for the help!
I finally got it to work. The problem was apparently the reagent itself. When I got a new tube and did the EXACT same procedure, the transfection worked!!
Thanks again for the suggestions! 
Yes, when we use it we avoid exposure to air as much as we can and you need to follow the protocol exactly. It should work.
Bye
thanks for the help!
I finally got it to work. The problem was apparently the reagent itself. When I got a new tube and did the EXACT same procedure, the transfection worked!!
Thanks again for the suggestions!
Hey guys,
anwar is right regarding storage...
I also had problems when I received a small aliquot of Fugene6 from a collegue the first time- it was stored frozen in an eppendorf tube. Then I read on the roche-applied-science homepage the following (see below). Sometimes it would be better to first read the pack insert, then start working...
"Both the new version of FuGENE 6 Transfection Reagent and FuGENE® HD Transfection Reagent must not be stored below 0°C! Further, to achieve optimum stability of the products the vials have always to be stored very tightly closed. Never aliquot the contents for storage!
Note: As a function of time, storage at low temperatures will separate active components of FuGENE 6 Transfection Reagent. Nevertheless, this effect is reversible upon warming reagent to ambient temperature and thoroughly mixing. Storage at low temperatures is not required to maintain shelf life.
• For FuGENE HD Transfection Reagent the order of pipetting must not be changed: Always dilute the reagent by pipetting directly into serum-free medium containing DNA. Do not allow the FuGENE HD Transfection Reagent to contact the plastic walls of the tube containing the serum-free medium during the dilution step."