does phenol:chloroform extracted plasmid creats problem in Mammalian cell cultur - (May/27/2006 )

hello everybody, I am using pcDNA3.1 vector for expression studies in Mammalian cells, and i wanna know whether phenol:chloroform extracted Plasmid can creat any hinderance during this or plasmid without RNase treatment can ??????
thanks

It all comes down to how "clean" your extraction is. If you perform phenol chloroform and later on transfect your cells and you're not getting the expected results, try checking OD260/280 ratio. Maybe you're DNA is contaminated still?

Apart from that:try including a positive control transfection with (for instance) a EGFP reporter plasmid. Should give you some indication on how effeicient your transfection procedure is.

When I transfect cells, I mostly precipitate it and I make sure there's no residual Ethanol left by drying extensively.

You definitely want to extract with chloroform at least once, and preferably more than once, after the phenol extraction. This is to remove residual phenol. Extracting with ether also works, but this is usually less available, and has issues with peroxide safety.

Extract a second time (and possibly a third) with chloroform or chloroform:isoamyl alcohol to remove phenol.