transfection of HeLa cells - (Aug/05/2006 )

has anybody used Lipo2000 for transfecting HeLa cells? what shd be the starting confluency, >90% or 75-80% ?

what plasmid isolation kit would be best for transfection ?
(i mostly get a O.D 260/280 ratio of 1.4 )

After 48hrs, i do SEAP assay and i get very high background signal even in my untransfected cells and so the transfection is not working at alas i dont see any increse in fluorescence signal with my positice controls.

looking for some suggestions..

thanks,
jhilmil

75-90% would be ok, although higher cell density (90%) reduces toxicity to certain degree. We use Qiagen kit and OD260/280 ratio should be close to 1.8. You need to optimize transfection condition by using a fixed amount of DNA and varied amounts of Lipo 2000. For 96 well plates, 0.1-0.2 ug per well DNA and 1ug DNA/2-5 ul lipo ratio is somewhere for you to start with.

Do you have cat # for that? Thanks.