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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 :)

-klanarong-

Try increasing to 24 Ál 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?

-madrius1-

Hi klanarong,

Can you please briefly descried your transfection protocol please?

-stylothecancer-

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

-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-

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

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 :lol:



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.

-stylothecancer-