how do you improve transfection efficiency? really puzzled!

[Ian A. York]

In article <1122134629.145018.37030 at f14g2000cwb.googlegroups.com>,
bb <chris_blc at hotmail.com> wrote:
>Hi, I've got problems with human cell lines transfections (e.g. HEK293,
>SKNSH...etc). In my lab, we use Lipofectamine 2000, and Fugene 6. We've
>tried several combinations of DNA concentration and transfection
>reagent, but the max transfection effeciency is only about 30% (usu.
>~10%)!!! We did all steps according to the reagent manuals, but the
>results are just very different from what we heard. I'd be really
>appreciated if somebody can provide any hint on how to get a
>transfection with a >50% efficiency....thanks a lot!

There's no simple answer, and many cell lines are simple not all that 
transfectable -- with several of our lines we're pretty happy with 10% (or 
less) efficiency.  You've already tried DNA concentrations and 
transfection reagents: try other reagents; in my hands Lipofectamine 2000 
is not all that great, though I know with other cell types it can be 
excellent.  There are many reagents, and sales reps will often give you 
sample sizes if you're planning on using a bunch of it.  You can also try 
different cell density, which can sometimes make a very large change in 
efficiency.  You should also be very sure there's no mycoplasma 
contamination, which in our lab once led to an overnight reduction in 
efficiency from >75% to ~5% efficiency -- treatment with ciprofloxacin 
reversed the problem almost overnight, but it took us a month or two to 
figure out what had gone wrong.  

Good luck.

Ian 


-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England