PCR inhibitors in insect tissue?

Andrew Cockburn afc at gnv.ifas.ufl.edu
Tue Sep 6 10:25:14 EST 1994


In article <aquilla.1128645356A at sadye.emba.uvm.edu>, aquilla at salus.med.uvm.edu (Tracy Aquilla) writes:
> In Article <23AUG94.16244964 at skyfox.usask.ca>, edwardsg at skyfox.usask.ca wrote:
>>Has anyone else experienced problems with PCR inhibitors when attempting to
> ampl
>>ify insect DNA.  I have tried GeneClean, Elutip-d columns, multiple
> phenol-chlor
>>oform treatments, however, some inhibitors remain and this really knocks down
> th
>>e sensitivity. If you have developed a new or old but inovative protocol please
>>let me in on it.
> 
> This probably depends a lot on which insect and which tissue. Some tissues
> are easier to get clean DNA from than others. For example, fat body
> generally has lots of insoulble material in it. You may also be having
> problems with chitin. What tissue and what insect are we talking about? What
> is the evidence you have for the presence of 'PCR inhibitors'? Are you sure
> the problem is with this specific DNA?
> 
> Tracy Aquilla, Ph.D.
> Molecular Physiology and Biophysics
> University of Vermont
> aquilla at salus.med.uvm.edu

We PCR mosquitoes all of the time.  We grind up the whole insect, boil it,
and use an aliquot for PCR.  No purification, no inhibitors.

By the way, we find that this actually works better than purified nucleic
acids- I suspect because the RNA is chewed up in the crude homogenate.

Andrew Cockburn
USDA



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