Need help with transient transfection - (Apr/10/2008 )

Dear all,
I've been having issues with performing transient transfection on SK-MES-1 cells using lipofectamine 2000.
Firstly i've been having cytotoxicity issues with this cell line and needless to say my transfection efficiency is pathetic.
Hence i was wondering if any of you kind folks out there have got any experience with transfecting this cell line.
Any help would be much appreciated. Thanks heaps in advance.

-weii02-

QUOTE (weii02 @ Apr 10 2008, 06:34 PM)

How do you know you have Cytotoxicity? what happens exactly after transfection to your cells?

I have never used lipofectamine but I find so many people having difficulties with that. I used to use Calcium Phosphate transfection. I never experienced any problem with that and the preparation was easy and very cost effective. my plasmids could enter my cells very easily.

you can also go for ROCHE's FuGene....I know it is a very good buffer.

-Aidin-

If there is too much lipofectamine compared to cells, this will induce toxicity. Try to use lower volumes of lipofectamine. Also make sure the DNA prep is clean and the maxiprep gives endotoxin free DNA.

I am not experienced with this cell line.

-scolix-

It will help if you do a titration with vaired dosages of lipo and one or two DNA dosages. Do it in 96 well plates to save your cells and reagents. a good starting point will be 0.25 to 0.6 ul per well with 0.1 ul increment and 0.1 or 0.3 ug DNA per well. Afterwards, you can scale up to cells in large wells based on the differece of surface area. good luck.

-genehunter-1-

I am not familiar with this type of cell but i know that to avoid the lipofectamine 2000 induced cytotoxicity you should plate your cells to be 90 to 95% before transfection .

-spanishflower-