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Thread: QPCR Contamination or Evaporation?

  1. #1 QPCR Contamination or Evaporation? 
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    I was running a QPCR with standards and 80 wells (40 samples) for a soil fungus which I expected to all be negative. The standards were as expected and 79 of the wells were negative but one well had very strong reaction. Amplifying about 15 cycles before the strongest standard then levelling off to half the height (y axis) of the standards. The other well filled with the same sample from the same tube was negative. I do work in the same room with the fungus I was testing for.

    Does this look like a contamination or an evaporation. I'm worried as evaporation from a well could damage the machine.

    Thanks for the help.


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    Forum Professor Zwolver's Avatar
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    Hi MikeS

    This sounds like a contamination. Occam's razor.. If there was evaporation, i doubt it would damage the machine. It would just be water vapor, and the machine is most likely made to resist this.

    However, to know, simply look at the volume in the positive cell. Is it less than the others?

    I never had positive result from evaporation, however it is possible in theory, that when concentrated too much, primers may anneal with one another, form bigger strands, and could possibly harbor a probe. This depends on the complexity of the probe. But you have to be very lucky to get this. Especially so early in Q-PCR.

    More logically, it would have totally evaporated and left a residue, and this residue may have fooled your detector.

    One question i do have... Why run Q-PCR with just suspected negative samples? Seems like an overkill to me.


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