Dear Philip H Jones ,

It is generally not possible to multiplex with an intercalating dye like SyBr green since you will not be able to discriminate the source of your flourescence (amplicon A or B). Multiplex assays always use mastermixes without any dye and use primers, probes or molecular beacons to discriminate between the two or more targets.

Best wishes

Soenke

As per Sönke Weinert's answer, it is inadvisable to use SYBR Green qPCR for multiplexing or differentiating samples with one reaction. Probe-based assays (TaqMan, etc) are much better suited to your needs. There are plenty of companies and websites that can help you create a probe-based assay that is capable of multiplex. The primary consideration is that you design probes that use different dyes (such as FAM and VIC/SUN) and won't dimerize to each-other.

Personally, I always get my primers and probes from IDT (Integrated DNA Technologies). The tools on their website are very useful and their application support team can help you design the assay if need be.

Now technically speaking, it is possible to use intercalating dyes (i.e. SYBR) for differentiation or multiplex, however it is much more complex and costly. One such method is called High-Resolution Melting (HRM) from Thermo Fisher using their MeltDoctor software. It requires a special calibration kit for your machine and a license for the analysis software.

Peter Chomczynski you can in principle do mulitplexing with Sybr green even a bit easier. If your TMs for the products do differ enough, e.g. 5°C you can acquire at two temperatures and with this you can see the deviate between both. Further as you said the melting would be an option. Here it is important to add that the difference in melting temp should be at least 5°C.

However, all of this are just workarounds, Sybr is intended for single plex with good primer design (no primer dimers) only