HELP: Transfection of Huh7 - (Feb/07/2008 )

Hi everyone!!

I have some problems to transfect Huh7 cells.
I am using pCDNA3-TAM67 plasmid DNA (AP1 dn) purified with midi prep kit (QIAGEN), but cells died.

My way to transfect plasmid DNA into Huh7 is the seguent:

1.Plate 2*10^5 cells /6well plate two days before the transfection .

2.6h before transfection I change media to DMEM - 10% FBS , no antibiotics

3. incubate 10ul of lipofectamine in 250ml Optimem for 5 minutes, and, during this time I mix 4 ug of plasmid DNA in 250ml Optimem

4. then I mix them gently and incubate for 20 minutes at RT

5. then I add mixture to cells

After O/N incubation, the cells were completely died.

Also MOCK cells (with only pCDNA) died, but Lipofectamine alone is not toxic

Why??
Were I wrong??

Thanks

I am not sure what is happening with your cells. I might guess something is not right with DNA but I am not sure.

For our transfections, we use 6ul of lipofectamine and 2ug of total DNA (maxi prep) for a single well in a 6 well plate.

Have you checked purity of your DNA? A260/A280 and A230?

Can you do a mock transfection using any other plasmid, maybe even extracted with a different kit?

Maybe these cells are extremely sensitive to endotoxins?

Yes, my DNA is more pure: A260/A280= 2,21

no: mock transfection is with pCDNA3 extract with the same kit.

It should be between 1.8 and 2.0. Browse the forum to see what might be contaminating your prep, there's severall topics on these ratio's.