Lipofection transfection of HEK293 cells unsuccessful - (Mar/01/2009 )

I have been having difficulty transfecting HEK293 cells by lipofection. My initial try yeilded very low proteine xpression levels. Subsequent tries yeild no protein production. Am at a loss, anybody can help?

Depending on the cell line, amount of DNA and amount of lipofectin used varying results in transfection effiicencies will occur.

Lipofectamine and other liposome based methods claim to offer up to 10% efficiency but i have never seen even close to this, maybe you could try electroporation as efficencies are much higher?

for 293 cells with optimization, you should get at least 50% transfection rate, although 90% is also possible with some luck.

I suggest you use EGFP or b-gal reporter plasmid to optimize the condition for transfection. What reagent are you using?

I agree with genehunter. 293 cells are among the easiest, if not the easiest cell line to transfect. Try optimizing te amount of DNA you put in, the DNA/Lipofection agent ratio, incubation time, etc.

madrius1 on Mar 2 2009, 03:55 PM said:

Hi, I was wondering if you transfect the 293 cells in suspension or adherent as I normally gain about 70-90% transfection using my protocol with cmv-gfp and saint-mix reagent.

I think i might know the problem in my transfection. I have been using plasmid from miniprep. I am new at this and didnt know that maxiprep should be used to prepare the plasmid as it is cleaner. Another possibility is that my DNA has degraded, but i have been storing them at 4degreesC which should be okay right?
Anyway I am gonna start all over again with fresh cells, fresh plasmid, fresh media.

I also wash the cells with PBS before transforming them. This possibily affects the lipofection right?

thanks alot u guys

MiniPrep or MaxiPrep doesn't matter in my case as long as the 260/280 ratio is above 1.5, but washing the cells could help your efficiency. Sometimes I wash the cells and transfect in serum-free medium as serum could be influence the efficiency, but in other cells (HeLa or CHO-K1) I transfect without changing the medium with very nice results. Good luck!

This is strange, but I have also had a difficult time getting my suspension HEK to transfect via lipofection.

Has anyone had luck transfection suspension HEKs?

Use linear PEI. There are some papers on that.

We use PEI for HEK293s all the time - it is much cheaper than the lipofection reagents and a lot less toxic

P