problems with transfection - transfection condition adjustment (Jun/30/2005 )

Hi everyone
I just did my first tranfections and I failed to have any positive cells. I'm transfecting a B cell line with pIRES vector with or without an insert.
For each transfection I used 1E7 cell in PBS with 20 ug of endotoxin-free plasmid DNA and ECM 830 electroporator. I apllied 330V. These are the conditions from a paper on this cell line. Any advice what I can modify?

Thanks a lot
Krzysztof

If you used liposome method, use PBS-free, antibiotic-free and serum-free medium or buffer during transfection. Lipo2000 gave high toxicity when longer exposure, be careful. Another reagent you may try is Gencarrier-1 which showed very low toxicity and high efficiency on WEHI-231, BAL17 and M12.4.1 lines.

I am using SuperFect to tranfect DNA into HUVEC cells. My question, however, regards how I can use the tranfected cells after transfection. I wish examine the ability of the tranfected cells to migrate in response to a chemotactic stimulus. Therefore, I would have to either transfect the cells on a migration filter prior to stimulating migration or split the cells after transfection. Does anone have any ideas which is best? Is it OK to split transiently transfected cells?
Thanks for helping.

Dear Krzysztof,

instead of buying in new transfection reagents why don't you try different electroporation conditions.

For instance, a) try increasing and decreasing the voltage, modify the pulse time, c) try a different electroporation solution (serum-free media, serum-containing media and PBS).

The other thing too, is how did your cells look after pulsing - were they dead or just not expressing your protein?? This info would help.


Good Luck either way,

AussieUSA.