So according to the below paragraph, I need to sequentially increase the concentration of the components. But how do I do that if I am using an already made maser mix kit with fixed concentrations from the company. I don't think we can even find out what concentrations are used for each reagent ?
Multiplex qPCR Assay OptimizationIf a multiplex reaction is not optimized, the amplification of a more efficient or more abundant target can inhibit that of a less efficient or less abundant target. This occurs because reaction components such as DNA polymerase, dNTPs, and MgCl2 become limiting in later cycles, and the amplification of the less efficient or less abundant target is compromised. Inhibition of a target's amplification will cause its Cq to be delayed in a multiplex qPCR assay relative to that in the singleplex assay. One method for optimizing multiplex reactions involves sequentially increasing the concentrations of DNA polymerase, dNTPs, and MgCl2.
Edited by kabtq9s, 11 March 2015 - 10:20 AM.