Fluorescence is the problem

It turns out that the problem with my real time PCR run on October 13 is something to do with fluorescence. In the last post I described how I didn't see any amplification, but the fluorescence levels appeared abnormally high from start to finish. Today I ran the PCR products out on a gel and found that the PCR worked very well, as expected with my well-used primer sets. The products were clean and lacked any primer dimers. So, if the PCR worked, it is either the fluorophores in the reaction mix that are bad or the machine is broken and doesn't detect them properly.

There are two fluorophores in the reaction mix we use: SYBR and ROX (newer machines don't use ROX). SYBR is the dye that binds to DNA and is used to track amplicon abundance between PCR cycles. Rox, on the other hand, is a passive dye that doesn't interact with anything the reaction. ROX and SYBR have non-overlapping excitation and emission spectra. Thus, the machine can idenpendently read the SYBR and ROX signals: the SYBR signal is expected to vary dramtically between wells and change throughout the PCR run while the stable ROX signal is used to correct for variance that arises because of differences in transparency between wells.

My next step is to compare our old Master Mix with the new suspect batch. I may run this experiment on the new machine next door to make sure it is not our old machine that is the problem. However, this requires learning to use a new machine and analysis software and burrowing PCR tubes from the machine's owner.