I've been running qPCR with Quantitect primers from QIAGEN. I've tried these primers a couple of weeks ago, and they worked great. However, I tried them again today (new cDNA, other reagents are the same as previous experiment), and basically everything amplified around cycle 30 (whereas before, I got amplification between cycles 19-25, depending on which gene I'm looking at). My triplicates are also terrible (difference in deltaCt up to 2 of 3 cycles sometimes), whereas they used to be quite good.
I think it's possibly a problem with my new cDNA, or it might be primer degradation (I resuspended them in sterile water instead of TE by mistake). Anyone got similar problems before?