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Fanatic_scientist

Optimal primer concentration qPCR

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Hello,

In qPCR, is there an optimal concentration for primers with a total volume of 20ul per reaction?

Thanks for your answer in advance!

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What qPCR kit (or just enzyme) do you use? It depends on both the primers in question (how easily do they form primer-dimers) and the enzyme used, however I generally used 250 nM (for both the forward and reverse primers). Optimal primer concentration can only really be obtained by titration, but I have only done this (once) when initial results were dubious/contained primer-dimer peaks (increasing annealing temperature wasn't really viable/did not help either). When titrating, Thermofisher (Sybr Green) recommends checking the 100-500 nM primer range, however other researchers sometimes check between 50 nM and 900 nM (as of researchgate). 

https://www.thermofisher.com/nl/en/home/references/protocols/nucleic-acid-amplification-and-expression-profiling/pcr-protocol/sybr-greener-qpcr-supermix-universal.html

 

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