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PCR unspecific amplification - (May/29/2007 )

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Don't u hate the "magic bands!" angry.gif So, if you are extracting your water samples simultaneously then it should not be environmental contamination in the DNA extraction room because you should see this in your extraction water control. It shouldn't be random things in the master mix because you don't see it in the water control for the master mix. This does narrow things down to the template, as others have pointed out... This brings me to two questions. First, is the template for the positive control reactions a pure/enriched template (ie: plasmid or previously positive PCR?) Second, and somewhat dependent upon the first answer, I am also questioning your primers, perhaps it is time to buy new stocks?? How long have you stored your primers etc? I guess the theory is that perhaps you are getting non-specific amplifications in the more complex template environment, but your primers still look good on the positive control because it is a purified template?

(assuming purified positive is used, and maybe I am off base with this plan -- Comments anyone???)
Possibly one way to test this theory would be to spike the purified positive control into the other samples, if the problem is non-specific amplification because the sample is not positive (or degradation of the sample has broken the template between your primers) then you will not see the extra bands when the appropriate template is present, however if you still see both sets of bands (especially at the same or similar concentrations) then maybe something is wrong with the primers??


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