Sponges

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Tue Jul 7 21:46:41 EST 1998


In article <35A223A1.BE07A05E at uni-koeln.de>, akd38
<Holger.Stalz at uni-koeln.de> wrote:

> I'm looking for a method of isolating  genomic DNA from the siliceous
> sponge Geodia cydonium. I tried the QIAGEN Blood & Cell culture Kit but
> wasn't succesful at all.
> The main problem is caused by the silica pieces which I achieved after
> producing single cells and the gemmulae which couldn't be lysed due to
> the membrane associated silica pieces.
> If anyone had ever heard of someone who had ever lysed any kind of
> membranes of siliceous sponges, it would be nice if YOU could inform
> me...

Was the problem caused by binding of the DNA to the silica?
If so you may be able to elute it quite cleanly as this is
similar to the basis for the methodology for kits like Promega's
Wizard.  There the method is to adsorb the DNA in high salt under
denaturing conditions and then elute with low salt buffer (or
water).  I don't know how the QIAGEN kit works but if it is by
ion exchange this would not be compatible.
     So, if binding to the silica is bad (could cause shearing)
then try and keep the ionic strength low to limit this.  If
shearing is not too much of a problem then adapt the silica gel
purification methods.  One "gotcha" is that although silica gel
allows purification away from most proteins it may result in
DNase contamination (this was a major problem with Promega's
early kits) so you may need to clean up the DNA (eg. proteinase K
or phenol extraction).
     I hope that this gives some clues,
          Bernard

PS: I strongly suggest you try posting to bionet.molbio.methds-reagnts
as this is just the sort of question the group thrives on.
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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