From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!agate!library.ucla.edu!europa.chnt.gtegsc.com!howland.reston.ans.net!cs.utexas.edu!uunet!newsfeed.pitt.edu!quadra800.pathology.pitt.edu!user From: rapr@med.pitt.edu (Robert Preston) Newsgroups: bionet.diagnostics Subject: PCR Inhibition Water Chloramines Date: Mon, 27 Mar 1995 12:40:10 -0500 Organization: U.Pittsburgh Sch. Med. Lines: 39 Message-ID: NNTP-Posting-Host: quadra800.pathology.pitt.edu > > 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@med.pitt.edu University of Pittsburgh Pittsburgh PA 15261 .