PCR Inhibition Water Chloramines

Robert Preston rapr at med.pitt.edu
Tue Mar 28 08:33:30 EST 1995


I am forwarding a message I sent to bionet.diagnostics, since the info on
possible chloramines in lab water supplies is not common knowledge and MIGHT be
a problem in PCR-inhibition situations as in the thread-fragment below:
> 
> > > 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.  
> 
 Depending on the source of the water, it is possible for it to contain high
 levels of chlorinated nitrogenous compounds that neither show up on
 conductivity meters nor are removed by distillation.  In fact,
 distillation actually CONCENTRATES monochloramine in the distillate
 by a steam distillation mechanism: at one place, the distilled water
 reservoir for a departmental supply smelled a bit like a chlorinated swimming 
 pool.  Over several days time, monochloramine in a "purified" water reservoir
 reacts with itself, catalyzed by acidity from contamination by airborne CO2, 
 to make dichloramine in varying amounts.  Varying (micromolar) levels of 
 Dichloramine were the culprit in one of our irreproducible enzyme assays.
 (we did not, initially, think that the water could possibly be impure, since
 we had two glass stills operated in series in our lab, with departmental 
 distilled water as the feed.)
 
 FRESH charcoal filtration during purification of the water
 prevents accumulation of chloramines in a purified water reservoir,  
 but if the local municipal purification is chloramine-based rather than
 chlorine-based, a charcoal filter can be saturated in a relatively short time.
 
 Chloramine-related problems tend to be highly sporadic and irreproducible
 because of the dynamics of the many variables involved.  The simplest way to
 test for the material (at least, for concentrations that would be causing 
 problems) is to use the starch+iodine test for chlorine that any high-
 school chemistry student should know.  It also has a weak UV absorption peak
 around 245 nm, as I recall, which can be used to detect the material (at
 least, in a background of "pure" water) if 10 cm pathlength cuvets are 
 available (and truly pure water is in the reference cuvet!).



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