Greetings. I'm running a TaqMan assay on the StepOnePlus R-T PCR System, using the TaqMan Fast Advanced Master Mix. I validated an assay that gives me very precise Ct values for positive controls (~21). However, on two non-consecutive runs this past month, for the same controls, using the same reagent lots and reaction conditions, the instrument has given me Ct values of ~28, which is about 100-fold change.
Passive reference signal intensity is no different than with other successful runs. The only QC flags I'm seeing are No Amplification and Exponential Algorithm Failed, which I regularly see for negative samples. Any help on this matter would be appreciated. Thank you.
Thank you for your responses. Andrey, it is unlikely the polymerase became inactivated because I started using different master mix aliquots when I first noticed the problem. Ct still oscillated, despite using a new aliquot.
Jack, my control is a ~90 bp dsDNA fragment. The TaqMan primers flank this sequence. Again, I use a new aliquot with every new run, so this effect is also not expected.
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