hi all-
it might be a very basic question but i struggle with the rational idea in my head-
when performing qPCR we need F primer, R primer and a probe (taq-based qPCR).
i usually use the same concentration for all molecules (1ul of 5uM in final 20ul rxn vol ending up in 0.25uM in rxn)- both primers and probe.
recently i have realized some protocols suggest to use double volume / stock concentration of primers (2ul of 5uM per rxnor 1ul of 10uM stock, to reach 0.5uM for EACH primer in rxn), while probe final concentration in reaction remains 0.25uM.
is this something you all follow? have I made all my assays the wrong way so far? does this mean I could have got better results all this time?
can anyone explain whats the rational principle behind it?
thank you all
Hello Miri Hillel
Usually, it is recommended that a 1:1 primer-to-probe ratio be used for highly expressed targets. On the other hand, for targets expressed at lower levels you may increase the primer-to-probe ratio. So, accordingly you will have to adjust the primer concentrations to obtain optimal results.
Best.
Hi,
the amount of Primers is steering the PCR efficiency, so when you tritrate your oligos you will see that such a ration is mostly more efficient. In addition, you don't need much more of Probe from reaction kinetics
Best Sven
- Badges
- Science method