disappearing PCR product

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Thu Jun 17 10:21:59 EST 1993


In article <1993Jun15.145544.21902 at gserv1.dl.ac.uk>, CS at abc.univie.ac.at ("Christoph Schller") writes:
> Hi
> We frequently (with different batches enzyme) observe the
> disappearing of PCR products if they are EtOH prec. O/N on -20.
> Has anybody else observed that? - And how to get rid of this
> mysterious nuclease (phenol doesn't work).
> Thanks
> Christoph S.

IMHO, your problem is with precipitation, not nucleases.  No enzyme is
going to degrade ethanol precipitated nucleic acid.

To begin with, precipitate your DNA at +4C, not -20C.  It is well 
known (except by my graduate students) that DNA precipitates much more
effectively at the higher temperature.

Second, centrifuge the ethanol precipitate at a higher speed or for a longer 
time.  This is important if you have a relatively small amount of product.
30 minutes at 12,000 RPM is probably good.

Finally, add some carrier, either nucleic acid or protein.

Andrew Cockburn



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