Real Time Contamination/Background Issues - (Aug/24/2006 )
I am doing Real Time PCR from genomic DNA with a Taqman probe that is labeled with a Famra reporter and a Tamra quencher. The passive reference dye is ROX. The NTC and the first few standards on my standard curve consistently amplify with a Ct of 25. The higher standards have a lower Ct. At first I thought that this was caused by contamination, so I cleaned all supplies, changed all reagents (including new dilutions of primers and probe), and began setting up my plates in the hood. This did not solve the problem, and I thought perhaps the probes (which are over a year old) were degrading during cycling. I ran wells containing only probe, ROX, and water. Those wells showed a Ct of 25, but on the multicomponent view, the FAM actually decreases over time while the ROX and TAMRA remain relatively stable. I don't know what that means for the probe, and I can't figure out why decreasing FAM would be interpreted as amplification by the software program. Any thoughts or suggestions would be appreciated.
seriously, that's what I would do. that sounds like a real mess. I bet you could spend tons of time fiddling with it and not get it to produce reliable data for you (and if you have to fiddle too much, is the data reliable anyhow?)