How to standardize Real-time duplex PCR using SYBR green master mix?
I'm standardizing a real-time duplex PCR using Power Sybr green master mix (Applied biosystems). The Tm values of my 2 amplicons; 123 bp and 213 bp are 70 and 75 C respectively. The primers have 99% and 99.2% efficiency when calculated by standard curve method. Simplex PCRs are fine with crisp peaks appearing after melt curve analysis (0.5 C/s ramping). When tried in duplex format only the peak corresponding to 70 C is appearing and agarose gel also showed presence of 123 bp band. Increasing the concentration of primers corresponding to 213 bp product or introducing MgCl2 into the PCR mix did not help at all. Is there any method to standardize duplex PCR using Sybr green? Any thoughts?