i am currently making assay of syber green based real time pcr for detecting rare isoforms.To make them specific ,can i use say 1 p mole of forward and 5 p mole of reverse primer,technically for taqman based assay this is valid then it should be valid for this also ,so long as efficiency is 95 % above we should be able to use ,i want any opinion
Yes you can do this for sure. In an ideal situation you would test a whole range of concentrations of both primers in combination to find the most efficient assay.
Yes you can do this for sure. In an ideal situation you would test a whole range of concentrations of both primers in combination to find the most efficient assay.
Yes you can do, depending on your assays. I always used the same concentrations for forward and reverse as my protocol was already optimised. I Will suggest to search on the literature similar detections as yours and see what is referred, maybe you can find a specific concentration snd then try yourself two up and two down if that makes sense so you can have sort of a good range of data to compare with the efficiency of the reaction and the r2 of the curve.
Yes that's is fine. Test a panel of primers mixing and matching forward and reverse from 50nM to 250 nM
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