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samples come at same Ct as NTC - contamination impossible (Sep/10/2008 )


yesterday i observed a very strange thing with my pcr. this taqman pcr workes every time very accurate (NTC=undetermined) and samples come at Ct~30. but now my ntc is also coming at Ct~30. i tried to pipette the same pcr with all solutions fresh and pipette tips with filters (i normally not use). but i observed the same shit.

second my SYBR pcr, which i also tried, showed ugly dissociation curve (usually at 85°C)

what could be the reason for this observations?

thanks for suggestions


I've seen something like this a few times. I assume your NTC just creeps above the threshold and it doesn't look like a real positive.

In this case there may be some degredation of your probe.

In one lab, the ordered new probe and primers and this solved the problem. When a similar situation occurred in our lab and we ordered new primers and probe, the issue was not corrected. We played with the concentration of probe (lowering it) and this seemed to help a bit.

Good luck.