Remove endotoxin?

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Fri Aug 21 08:55:00 EST 1998


In article <6rimvv$pfp$2 at news1.bu.edu>,
  rocket1 at bu.edu (Richard Near) wrote:
> I have recently had low efficiency transfections using plamids made
> by alkaline lysis /phenol extraction/ and PEG precipitation. I saw
> a blurb from Quagen suggesting that endotoxin contamination may be
> a significant contributor to such low efficiency. I grow my plasmids
> in end- bugs. Aside from this (and purchasing a relatively expensive
> kit ) , is there a (or some) ways to remove endotoxin??
>
> Rick
> rocket1 at bu.edu
>
> Thanks
>
>

end- usually means endonuclease negative, not endotoxin negative.  Aside from
growing a plasmid in gram positive bacteria (which might require a different
replicon) I don't see how switching strains would reduce LPS contamination.

I have seen the same Qiagen literature you are refering to.  The levels of
endotoxin in their "ordinary" plasmid kit seem comparable to 2X CsCl; I have
used both methods in the past and found the transfection efficiency to be
similar (I've never done "side-by-side" comparison, just my impression).  I
would probably only use the endotoxin-free kit if I was looking specifically
at an inflammatory response (e.g., mapping an LPS-inducible promoter element,
or something like that).

Are you doing anything to get rid of the PEG? I don't know about transfection,
but residual PEG can inhibit a lot of biological processes.

Nick Thedorakis

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