My dulition target is 10 pmol for primers. I dilute primers without any problem at qpcr, multiplex pcr and classic pcr. Sometimes, I diluted probes and it didn't work. What is the least/minimum concentration for probes to be processed?
Why do you want to dilute your primers/probes to the minimal effective concentration? The goal of optimising a QPCR reaction is to get the best possible efficiency for a particular reaction which will depend not only on primer/probe sequences, but also on the QPCR reagents you use, annealing temperature in PCR cycle, target cDNA concentration etc. etc. In my experience, using Agilent Brilliant III ultra-fast reagents and a 2 step amplification protocol, QPCR reactions to detect rare transcripts from limited amounts of cDNA work optimally with primers at around 200 nM and probes at around 500 nM. For a conventional 3 step amplification protocol with most other QPCR mastermixes these optimal concentrations fall to around 150 nM for primers and 400 nM for probes.
Some reactions need more than 10 pmol. For classic PCR, we add 20 pmol of primers.
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