TaqMan dual-labelled probe low fluorescence problems - (Oct/10/2007 )
I work in a diagnostic lab and have been given the task of developing a test based on real-time PCR. I have taken over from another employee and am trying to figure out where she left off.
My major problem is that using the same reagents, PCR conditions, analysis etc as has previsouly been used, I am getting very poor data. Most notably the level of fluorescence is about 10-15 fold lower than on runs performed by this previous employee, and the Ct values are somewhat larger.
Any ideas on what can cause Fl to drop like this? Primers/probes are in date and stored according to directions.
Any help would be enormously appreciated.
ps We are using ABI reagents and Corbett rotorgene cycler+software.
maybe there is something wrong with your sample RNA or cDNA?
your instrument settings are also the same? for example, the sensitivity of the detector (you can change this on the rotorgene cycler).
I think your probe is not very well labeled.You can label it again from other supplier.