chrisb at hgu.mrc.ac.uk
Fri Aug 21 06:03:14 EST 1998
Richard Near (rocket1 at bu.edu) 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??
Of course -- use a Qiagen kit to make your plasmid! Why do you think
they mention it as a problem? Alternatively, double banding on CsCl
removes endotoxin equally well.
But you should check that it really is not just a DNA problem. Try
mixing your DNA with another batch of DNA you know to be of high
quality (ie highly efficient at transfection). If endotoxin (or any
other diffusible contaminant in the DNA prep) is the problem, there
will be trans-inhibition of transfection in the mixture. If the
transfection efficiency is largely unchanged, the problem is in the DNA
itself, which might have been damaged by phenol or UV.
Chris Boyd | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of / Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \ Edinburgh, EH4 2XU, SCOTLAND
More information about the Methods