destroying dna with DEPC

suter at VAX.MPIZ-KOELN.MPG.dbp.de suter at VAX.MPIZ-KOELN.MPG.dbp.de
Wed Dec 23 08:56:46 EST 1992


Subj:	? How to destroy DNA chemically (to reuse DNA columns)
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+Well, the old cheapskate (me) has broken down and bought a bunch of
+silica-based columns for DNA column purification (plasmid).  We went with
+those from Machery Nagel (Nucleobond) bought in the US from The Nest Group.
+These are similar to the Qiagen columns, but the Nest Technical support was
+more informative and they were a little cheaper.++
++
+They work fine, as many of you have attested previously.
+
+They are expensive, though, and I'm toying with how to re-use them.  The
+critical element is to avoid pH above 9 for long periods (in minutes, I'm
+told). The important aspects would be to 1) retain binding ability and 2)
+remove DNA that might contribute to the next prep.
+
+We tried washing the column with DNAse solution (200 ug/ml in Mg++
+containing buffer, RT for 60 minutes) and re-stripping.  When we
+re-stripped again, though, we recovered plasmid "contaminant" that amounted
+to about 10e-5 of the original amount, based on amp-containing transfection
+units.  I think this is too much to allow re-use.+
+
+I'm thinking now about *chemical* methods of degrading/destroying the DNA
+bound to the column.  1M NaOH is out (that destroys the silica column) and
+I'm not too keen on 4 M HCl for the same reason.  I guess I'm leaning
+towards an alkylation agent, maybe Acetic anhydride, though I really don't
+have a good feel for what might work best. Maybe an oxidative reagent, like
+peracetic acid.
+
+So... any chemists out there?  What should we try that works near neutral
+pH in aqueous or anhydrous conditions that should destroy the biological
+activity of the residual DNA, that is safe and relatively cheap?  If you
+have reasonable suggestions, I'll try it out and post a followup.+
+
+dennis

i use DEPC (diethylpyrocarbonate) which is an alkylating agent, to 'inactivate'
DNA or RNA present in pipettes or bottles or whatever. (the nucleic acid does
not disappear, it simply is modified in a biological inactive compound). this
treatment helps me to overcome PCR contaminations, so i presume it is very
thorough. 
generally, i mix 2 microliters vigurously with 50 ml H2O. Apply to contaminated
object (fi: suck up into pipet a couple of times, rinse bottle, apply to
column (oli dT cellulose to prepare mRNA)). Remove DEPC either by letting dry
well, or rinsing with abundant amounts of H2O or other (non TRIS containing)
buffers (you shouldn't be able to smell it anymore, however, this is difficult
to achieve.

DEPC is a mutagen !!!!! only use it in the hood !!!!



clemens

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Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter at vax.mpiz-koeln.mpg.dbp.de
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