(none)

John McDonald mcdonald at pop.nih.gov
Thu Mar 2 18:12:05 EST 1995


Hello all,

   My question concerns winston-prep DNA, mitochondria and PCR.  I have
been doing PCR with degenerate primers and have a problem with some kind of
contaminant.  This contaminant is of approximately the right size (340 bp)
but when used as a probe for a southern does not light up any yeast
sequences.  Nor does it light up any sequences from the most likely
contaminant, E. coli.  This band does not show up in no-DNA control
reactions.  I have used two commercially prepared yeast DNA preps from
different companies and both give this band.  Also, both preps were
contaminanted with E. coli DNA (southern blot with different PCR products
as probes lit up E. coli sequences).  I have made two of my own
winston-preps and still have this band from outerspace (confirmed by blank
southerns).  One of the winston-preps was made with solutions that were run
through microcon filters to remove DNA larger than 200 bp and the yeast
cells were grown in synthetic complete medium.  Although I still can not
completely rule out other sources of contamination, I think that this
sequence maybe from yeast.  Perhaps it is from mitochondrial DNA or a
DNA/RNA virus.  Does anyone have information about whether mitochondrial
DNA or viruses are isolated in a winston-prep and if so are they in
sufficient quantity to be detected on a southern (~1 microgram genomic
DNA/lane).  Any other reasonable explanations that people can offer would
be appriciated.  Thanks.

John McDonald





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