john.brennand at gbapr.zeneca.com
Mon Apr 15 10:52:36 EST 1996
My 2p worth
A properly done miniprep shouldn't contain any genomic DNA. So (you
dont specify) is the smear just normally digested genomic DNA that is
obscuring any plasmid bands ?
In any case I would make solutions I,II,III up fresh and try again.
What happens if you incubate the miniprep (no additions) at 37o for an
hour and then run the gel ? If it degrades then you have DNAse
contamination that is only being activated upon incubation at higher
temp - if that is the case, phenol or Strataclean the minipreps.
Does DNAse need salt to work ? ie, if you add salt (buffer) to your
miniprep and incubate at 37o does that accelerate degradation ?
If you are losing the plasmid DNA only upon incubation with an enzyme, I
would suspect contamination of the enzymes - or more likely, the
incubation buffer, water, tubes, tips, etc.
Hope this helps.
PS like most people, we now use kits by Promega, Qiagen or Bio-Rad and
have never had any problems - a good investment compared to the cost of
people's time in trying to sort things like this out.
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