Hello everybody.

I have performed efficiency tests for primers of 12 genes with UPL probes in a Lightcycler 480 to obtain the curves with 4 dilutions of cDNA. I obtained optimal results for 10 genes, but the remaining two did not amplify. When I calculated the 2nd derivative, the wells of these genes marked green (negative) or Blue (uncertain).

I don't know what could have happend:

1. I use the same cDNA stock

2. The same protocol settings in the Lightcycler

3. One plate (96 wells)

The only thing I believe is that the primers didn't work or the probe is expired (they are stored at -20ºC protected from the light...but they were bought a long time ago (I think 3 or 4 years ago).

I hope someone can give me some ideas!

Thanks!