transfection problem: DNA purity

Bruno Cenni bruno.removethis.cenni at removethis.insel.ch
Mon Nov 19 02:11:57 EST 2001


Hi Ricky,

unless you use very sensititve or special cells (eg. primary) endotoxins
don't matter much. Standard cell lines like CHO, HeLa, COS, HEK293, HepG2
etc. trnasfect easily and well with CaPO4 or liposomes using plasmids
prepped with normal Qiagen or Promega or other protocols (ie not their
endofree variants) . However purity matters generally, as well as
degradation (eg. exposing the DNA too long to the first lysis buffer that
generally contain NaOH). Another concern is losing a plasmid by
recombination or contamination...

Bruno


"Ricky Leung" <b883732 at logic.itsc.cuhk.edu.hk> wrote in message
news:9t8hsl$1ml$1 at justice.csc.cuhk.edu.hk...
> 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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