transfection problem: DNA purity

Jose Enrique Mejia jmejia at molbiol.ox.ac.uk
Sun Nov 18 12:57:28 EST 2001


Hi Ricky,

Did you run your plasmid prep on a gel to check the DNA was intact?

I wonder if  your Concert kit involves running your lysate through a column
and then an elution step. Some resins used in the columns are known to
release contaminants that interfere with enzymes or are toxic to cells.

Jose



Ricky Leung wrote:

> 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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