c******e
发帖数: 350 | 1 Sorry for no Chinese input at work.
I had some real-time PCR run with TaqMan probe. A false positive result was
showed for an actual negative sample. When I checked its amplification curve
(green line in figure below), I found it's different from other samples.
The signal intensity never goes up as a real amplification curve (of even a
negative sample). However, because the change of the intensity from baseline
occurred earlier, the Ct was calculated as lower than other negative
samples, and the software caught it as a positive sample.
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More Information Updated:
The Ct for the green curve samples got at 38. In another experiment a negative sample gave a Ct at 36.
These 2 occurred very rarely (2/50). When I repeated it, 1/4 chance gave negative result.
I wonder what would be reasons for this abnormal amplification? And how to
prevent it?
Thanks a lot! | R****n
发帖数: 708 | 2 Ct cutoff for real-time is 40. primer dimer will be a possible cause. You
shouldn't let it run that many cycles. | C*******e
发帖数: 4348 | | c******e
发帖数: 350 | 4 Thanks a lot! 但是TaqMan probe不是不会测到primer dimer么？
The Ct for the green curve samples got at 38. In another experiment a
negative sample gave a Ct at 36.
These 2 occurred very rarely (2/50). When I repeated it, 1/4 chance gave
negative result. |
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