transfection problem: DNA purity

Ricky Leung b883732 at logic.itsc.cuhk.edu.hk
Sun Nov 18 09:51:01 EST 2001


Hi everyone,

I've a problem during transfection. I tried to transfect pcDNA-lacZ to
mammalian cells using lipofectamine plus. When I used stock pcDNA-lacZ for
transfection, everything worked fine and b-gal was obviously detected by
western blot analysis. However, when I used pcDNA-lacZ which was prepared
from Concert miniprep kit, no b-gal was detected. Then I tried to further
purify the plasmid by phenol/chloroform extraction. The result was
improved a little bit. Beta-gal was marginally detected. Such poor
yield would be due to endotoxin, right? So, I wonder to know if there is a
efficient way to remove endotoxin. Any comments and suggestions are
welcome.

Thank you very much.
Ricky  




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