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PCR Inhibition Water Chloramines

Robert Preston rapr at med.pitt.edu
Mon Mar 27 12:40:10 EST 1995

> > 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.
Over several days time, the monochloramine in the "purified" water tank
reacts with itself, catalyzed by acidity due to CO2, to make dichloramine in
varying amounts.  FRESH charcoal filtration as a final step (after
distillation or deionization) removes this cantaminant (and any other
chloraminated material)
but if your municipal purification is chloramine-based rather than
chlorine-based, your charcoal filter will be saturated in a short time and the
problem will return.  If you have a chloramine-based municipal treatment
system, you need to know that fact and avoid using the water for reagents unless
you watch the charcoal filter situation religiously.

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 stuff (at least in the levels that would be causing problems)
is to use the starch+iodine+KI test for chlorine that any high-school
chemistry student should know.  It also has a UV absorption peak around
245, as I recall, which is weak but useful for detecting the material if
10 cm pathlength cuvets are available.

You should also be aware that autoclave steam supplies are not beyond suspicion
as sources of contamination, depending on how well-managed your steam
department is.

Robert Preston                                    rapr at med.pitt.edu
University of Pittsburgh                        Pittsburgh PA 15261

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