In article <6s6tfh$i50$1 at wnnews.sci.kun.nl>, "Gerrit Bouw" <gbouw at sci.kun.nl> wrote:
:Hi there,
::I am in desperate need for a transfection protocol which gives up to 60 or
:70% transfection
:efficiency in CV-1 and AtT20 cell lines. Are there such protocols anyway? I
:would be
:very happy to test different protocols on my cell lines...!
Sure. Try very harsh electroporation conditions killing 80% of all cells,
the rest will be nearly 100% transfected. Here we have achieved this
for PC12 and CHO. There is no reason why it won't work with CV1 (whcih
are easy to transfect) and At20 (whcih I never worked with). Key things to
keep in mind:
- use high cell concentration (3x10^7/ml)
- use high plasmid conc. (100 ug/ml)
- find in what buffer/medium cells survive best (1-2 mM amounts of MgATP
and/or 5-10 mM gluthation may be helpful)
- use as long impulses as possible (highest capacitor available, may also
try to decrease volume 2-fold)
- 6 mM butirate in the medium might help
- Dima
