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Co transfection with MCF-7 - help? (Jul/13/2005 )

I've been trying to optimise a transfection of MCF-7 cells with luciferase. I got it to work, but now, i'm trying to cotransfect with a luciferase and cat plasmid, and it aint working.
I'm using lipofectamine 2000, 1 ug total plasmid DNA, and a ratio of 1 : 0.5. This was working fine with just the luciferase, but now with the added CAT, it's all bad. sad.gif
Any thoughts?
Any ideas on what could be going wrong?



Could it be that your once-transfected MCF7 cells are now somewhat resistant to transfection? We have a similar problem with one of our artificial cell lines, where we cannot transfect again with another plasmid. If the artificial cell line did take up the new plasmid, it lost the first one. I've got no idea why that would happen since the first transfection was a stable the gene of interest should have been integrated. Why would it suddenly spit it out when re-transfected 4 years down the road?.

If anyone can provide a logical explanation to this, it will be appreciated!



Sometime lipo2000 causes problem in addition to toxicity for common cell lines, you are not alone.
Your protocol seems all right to me. All I can think of is promoter competition between Luc and CAT?
Also, one trick for co-transfection - always to make sure cells uptake one plasmid also uptake another one by adjust the molar ratio.

If it is still not working, try another reagent.
Our lab has changed to Gencarrier-1 because it is more consistent, effective and much cheaper. lipo2000 is not consistent and has toxicity issue when longer exposure, and fugene is not good for co-transfection and is well too expensive.


calcium-phosphate precipitate is efficient on this cell-line