DNA sterilization before transfection

CSC un691cs at genius.embnet.dkfz-heidelberg.de
Tue Aug 3 02:44:32 EST 1999


On 2 Aug 1999, Chris Boyd wrote:

> CSC (un691cs at genius.embnet.dkfz-heidelberg.de) wrote:

> : On 2 Aug 1999, Chris Boyd wrote:

> : > Um, what's wrong with incubating the DNA at 80 degrees or higher for an
> : > hour or so?  (This is a genuine question). Surely this will get rid of
> : > virtually everything alive but spores?  I can't think it would be worse
> : > than EtOH precipitation in this regard anyway.
> 
> : It will denature your DNA.
> 
> Yes, but not irreversibly. Supercoiled plasmid DNA (which I naughtily
> assumed was the form of Dr Gay's material) will renature properly after
> cooling -- otherwise the Holmes & Quigley boiling prep for plasmid DNA
> would be worthless. Similarly, linear DNA will reanneal, though less
> completely -- otherwise PCR would be worthless!

Oops ! I actually do NOT believe (all) DNA will denature during the 1
minute at 100C, as it is thigthly packed in histones and such. So you can
still recover enough supercoiled DNA from this miniprep (llok at your
gels: you will see a lot of linear and open circle as well).
On the other hand, if you want to do transfections, you need high quality
supercoiled DNA, not DNA which was denatured and then (partially)
renatured. I advice against heat sterilization of DNA used for
transfections.

Kind regards,

Clemens Suter-Crazzolara, PhD

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