PCR hell

Jeanne Loring jloring at genpharm.com
Mon Jan 23 17:01:26 EST 1995


        Reply to:   RE>>PCR hell
When the primers "go off", do they degrade, or just inexplicably inactivate? Is
there any way, besides making new primers, to find out if this is the problem?

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In article <9501201302.AA10829 at compbio3.med.wayne.edu>, 
jeff at COMPBIO2.MED.WAYNE.EDU (Jeff Kramer) wrote:
>Descriptions of PCR Hell deleted<

Hello Jeff,
It looks like you have ruled out everything else except the primers.  
Primers can die, especially if you resuspended in water (Our labs 
experience).  In our case the hospital engineers pulse the distilled water 
lines with acid periodically to get rid of nasty stuff.  This leaves us 
with very clean water lines but the pH can drop to 2.4!!  The nanopure 
system doesn't change the pH. About a year ago our PCR just stopped, we 
went through everything (conditions, reagents, etc.).  It turns out that 
our water had killed our primers and nucleotides that were made up during a
time of low pH time.  Now we resuspend our primers in 1 mM Tris 8.0 (yeah 
that's weak but we haven't had the problem again).  I'm just a bacteria 
type, but I vote for bad primers.  The net.PCR.gurus may have different 
opinions.

Best of luck,
Brad
===========================================================================
Brad Nicholson              |"If it worked the first time, it wouldn't be 
Department of Pathology     |	research."...Brad Nicholson
University of Utah          |	Live from behind the Zion Curtian.
Salt Lake City, UT 84132    | 
brad at corona.med.utah.edu    |
or: (801)-581-4365          | My opinions are solely my own.
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