problem with 293T cell co-transfection - (Jan/20/2010 )

I have been using Fugene HD for co-tranfection befor i did, i transfected each of plasmid the result show high efficency of transfection but when i do co-transfection with the both of plasmid the result is not work
I use ratio Fugene HD reagent to DNA in one plasmid 12 ul : 4 ul and use ratio Fugene HD reagent to DNA in co-tranfection 12 ul : 8 ul (4 ul of each plasmid)
please discussion Thanks lot

Try increasing to 24 µl of Fugene. You have to keep the 1:3 DNA:Fugene ration as much as possible.

Also, can you describe the protocol you're using?

Hi klanarong,

Can you please briefly descried your transfection protocol please?

You might find it worthwhile to look at the manufacturer's instructions for transfections. I know that they have a chart for optimization that you might find useful to figure out the optimal amount of Fugene to add.

Are you using Fugene HD or Fugene 6?


Labrat612

protocal for fugene HD co-transfection
1 i plate 293T cells 1x10^6 cells/35mmdish O/N
2 next day in the morning i will replace 10%FCS RPMI with optimem
3 prepare the co-transfection mix
-dilute 4 ug of each DNA (total 8 ul) with 100 ul optimem sligtly gently mix
-next add 12ul of fugene HD reagent to mixer gently mix
-then incubate at room tep 15-20 min
-i dorp the mixer to cell and incubate cells at co2 37C
-finally after 4 hrs i will replace with 10%FCS RPMI and incubate cells at co2 37C 2days and use to analze
thanks for all comment

klanarong on Jan 22 2010, 05:10 PM said:

Hi klanarong,

Your protocol looks fine. But these are some of the variations i did, hope it would be helpful to you. Normally i will transfect my cell in serum free medium/ low serum medium ( around 2%), not sure why, I get a better result out of it. And after transfection, i will leave the transfection mixture with the cell in serum free or low serum medium for 4 to 6 hrs and then add in serum to the final concentration of 10% without replacing the cell culture medium. I will only change the cell culture medium after overnight culture. Yet, not sure why, I get more than 95% success with this protocol, with various transfection agent such as fugene, lipofectamine, genejuice etc.