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how do you improve transfection efficiency? really puzzled!

how do you improve transfection efficiency? really puzzled! - Protocols and Methods Forum

how do you improve transfection efficiency? really puzzled! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 07-23-2005, 04:03 PM
bb
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Default how do you improve transfection efficiency? really puzzled!



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!
Chris

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Old 07-23-2005, 04:12 PM
Ian A. York
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Default how do you improve transfection efficiency? really puzzled!

In article <1122134629.145018.37030@f14g2000cwb.googlegroups. com>,
bb <[Only registered users see links. ]> wrote:

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 ([Only registered users see links. ]) <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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