how do you improve transfection efficiency? really puzzled!
Ian A. York
iayork at panix.com
Sat Jul 23 11:12:58 EST 2005
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.
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
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