If you had multiplex PCR with 2 probes with melting temps of 41 degrees and 53 degrees and could not change the target sequence how can you adjust the reaction to acheive a higher yield? I think if this were about primers, I'd decrease annealing/extension temps and increase primers and template concentration, but I don't know what to do about probes. I could maybe change probe lenth without affecting the target sequence.

I’m not sure about your terminology/details here! For starters 'probes' usually equal primers in a regular PCR reaction (unless you had internal 'probes' to the PCR'd amplicon). And if you are doing a multiplex reaction are the two temps referring to one probe set? Or do you mean the amplified PCR products?

When we are talking about optimal annealing temperatures in PCR we are usually referring to the annealing temp of the primers. This temp may not be the same as the Tm of the amplified product (differential ‘melt curves’ in multiplex reactions are useful in qPCR!). For one set of primers you would hope that the melting temperatures were reasonably closely matched - there are many primer design programs out there that can help you achieve this. Yield can be increased by a number of ways such as increasing cycles and altering salt (e.g., MgCl2) concentrations, both of which may lead to an increase in non-specific products. Extension times depend on the length(s) you are amplifying and the type of taq polymerase. Changing primer length would not usually affect product yield, but could alter G/C content enough to get a better primer pair Tm match, and optimizing (not necessarily increasing) dNTP, primer and template ratios certainly can affect results.

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