Hi everyone,
I am tryingt to use Taqman probes for qPCR of isoforms. The Taqman assay guide on the website mentions using an alliquot of a 20X reaction made from a stock of 60X or 40X. However, I don't see the reaction mixture having any such labellings, instead the kit contains forward primer, reverse primer and a Probe, thus my question is how do I make a working solution for reaction mixture, excluding the reaction master mix. Any help on setting up and making the reaction mixture will be greatly appreciated.
Hello, Darshan Panchariya
Under normal circumstances, you will be provided with Master Mix buffer solution. It is recommended that you call the salesperson or company after-sales service and ask them to reissue it to you.
When using TaqMan probes for qPCR with primers and probe provided separately, you need to manually prepare a 20X working assay mix, as commercial kits typically refer to this pre-mixed format. To make the 20X assay mix, combine forward and reverse primers each at 18 µM and the probe at 5 µM, typically using 90 µL of each primer and 25 µL of probe, then add nuclease-free water to bring the final volume to 500 µL. This results in a 20X mix containing 900 nM primers and 250 nM probe, which, when added to the qPCR reaction at 1 µL per 20 µL total volume, gives final concentrations of 450 nM primers and 125 nM probe ideal for most TaqMan-based assays. You would then set up your reaction using 10 µL of a 2X TaqMan master mix, 1 µL of the 20X assay mix, an appropriate volume of cDNA template, and nuclease-free water to reach the final reaction volume. This approach ensures consistency and optimal probe performance, especially when distinguishing between closely related isoforms.
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