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I have a HEX/BHQ-1 probe that sometimes exhibits baseline creeping:
We prepare a big batch of our mastermix (with primers/probes, dNTPS, Taq etc...) and freeze it in daily use aliquots and I have noticed that these 'creepy' background lines seem to increase in frequency as the age of the frozen mastermix increases.
I hypothesise that the probe is degrading (fluorophore getting away from quencher) over time when stored frozen as part of a mastermix, however the creepy lines only appear in certain wells.
Any ideas as to why the creepy lines only appear in some replicates?
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I recall that this issue (raising background slopes in old master mixes) had surfaced before on the Forums, so I'd say that yes, it can happen. I'm sorry though, I couldn't find the relevant topic.
It seems to me that the "creepy positives" are confidently not false positives though - I mean, there's no log amplification, only a slow increase of fluorescence over time. This considered, I would say that you still have room to adjust baseline and threshold values to correct for the creeping slopes, but are those positive curves the most concentrated samples you have? If they are, and you raised the threshold, then they'd come out at cycles 34 and 38 which is a bit on the far side...
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