Removal of PCR inhibitors

KIM dkim at hector.NMSU.Edu
Sat Nov 27 12:17:34 EST 1999


One possible source of inhibitor is the GnSCN itself.  If the salt
crystallizes during storage, it will have no opportunity to redissolve in
subsequent steps of the purification procedure.  The crystals would be
visually indistinguishable from the diatomaceous earth.  Upon elution, the
only low-salt, warm temperature stage, GnSCN crystals will dissolve into
the eluate, leaving you with a DNA prep containing small amounts of GnSCN:
a potent inhibitor of enzymatic activity.

This was a problem when Promega switched from the NaI-based "Magic
Miniprep" to the GnSCN-based "Wizard Prep" formula.  During this
transition, there were many reports in this group of uncuttable plasmid
preps, etc.  A contributor called Promega tech support, and was given the
above explanation.  The suggested workaround is to pre-incubate the GnSCN
solution at 42 C, or thereabouts, to redissolve any crystallized salts.

Now, if you are having trouble with inhibitors in electrophoretically-
purified DNA (i.e. agarose band purification), which usually involves a
warm incubation step, I don't have a clue  :)

Daniel




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