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Michael G. Kurilla mgk2r at UVA.PCMAIL.VIRGINIA.EDU
Wed Mar 22 15:52:14 EST 1995


On Mar 18,  9:19am, "Kevin O'Donnell" wrote:
> Subject: Re:
> <<Your message>>
> 
> .  We've tried laborious/exhaustive DNA extractions, carefully 
> > monitoring DNA concentration, ensuring the integrity etc. of our reagen=
> t 
> >stocks, but   nothing seems to get over the reproducibility problem. =
>   If there is 
> >anyone out there who has experienced similar problems 
> > we would  like to hear from you, believe me!
>  
> We have just been through exactly this problem!  A promising assay just =
> wasn't 
> giving reproducible results - it was driving us mad.  Eventually, we trac=
> ed it to 
> the *water*.  The water used was filtered and autoclaved but when run =
> side by 
> side with reactions using water from a different source it was clear that=
>  it was 
> indeed the culprit.  Why? We don't know.  It might be fluctuations in =
> the pH or 
> ionic concentration of the water source.  
> 


What type of PCR machine are you using? There have been reports of
reproducibility difficulties between various wells of the machine. I know
this was documented in the original Perkin-Elmer machine (there was an
article in Biotechniques about this). This was demonstrated by preparing a
master mix (4.8mls) and running 48 identical reactions in all the wells of
the machine. The variation from well to well was quite impressive.

With regards to the water, I would a little skeptical of pH or ionic solutes
if you are using water prepared from a source that bases purity on electrical
resistance of the water. We use a Millipore system that measures the ohms
(pure water is something like >18Mohm). All we do after that is purify the
water through a 0.22 micron filter and store in aliquots. One point to
consider is that these systems don't measure organics (unless they carry a
charge). The final filtration is simply to ensure elimination of bacterial
contamination.

> We've decided to splash out (sorry) on some bottled water in future.
> 
> This may well not be what's causing your problems of course.  For example=
> :
>      
> >Our latest ploy is to hang rosary  beeds on the
> > PCR machine and play Elvis Costello very loud, but this has met with =
> only 
> > limited success.
> 
> Your problem may well be the variation in quality of Elvis Costello album=
> s.  
> Have you been playing 'Goodbye Cruel World' on the days it hasn't worked =
> 
> and 'Imperial Bedroom' on the days that it has?
> 

I've been told that a horseshoe over the machine works even you don't believe
that it brings good luck.

Mike Kurilla  



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