Please help (primer/ probe final volume calculations) - (Mar/09/2015 )

I am setting up a multiplex qpcr experiment very similar to the one in the link below, except that I am only using 2 targets.

http://www.sigmaaldrich.com/technical-documents/protocols/biology/multiplex-qpcr.html

I have been staring at the screen for 2 hours and a half trying to figure out how they calculated 0.4 ul for the probe and primers in Table P6-14. I tried several calculations but was not successful. 

I would appreciate any help with these calculations. 

Thanks in advance 

What is the problem?

They say "Volume of Stock Primers/Probes to Blend for 200 nM Final Concentration in Each Reaction."

So you just know you need 200nM final concentration of each in the reaction. With two targets you don't need to make a stock at all I think, just use the final concentration in suitable volume.

But the calculation is simple, you only have to take into account the amount of the primer you are looking at, and take the rest as a dilluting agent, doesn't matter if it is another primer or water.

So, for Target 1 forward primer you put 10 ul of 100uM primer into final volume of 100 ul stock blend, that means you have a 10uM concentration of that primer now. You don't care about otehr primers in the mixture, just this one.

And you take 0.4ul of stock blend for 20 ul reaction which makes 200 nM final concentration of this primers in reaction.

And this is same for all the other primers/probes.