I am working on ddPCR assays to detect a particular gene. I can input a dilution series of gBlocks from about 10^5 copies down to 10 copies per reaction. This input can be determined based on Qubit of the gBlock, or using the information provided by IDT (fmol per tube etc). After running the ddPCR assay, the quantified results by ddPCR come back the same order of magnitude. But not within 10-20% of the calculated input. As an example, if I put in say, 6,000 copies, it might come back as 4,000 copies detected.
What can I do to improve the assay precision further?
Hi Nathan,
It is not abnormal that there is a discrepancy between ddPCR and fluorometry/spectophotometry-based methods. The number you receive from IDT is just based on an OD-measurement and is not very accurate. Also, measurements with these fluorescent dyes can be off from the real value, as they rely on a standard (which can have quite some variation from lot to lot).
In general, these fluorescent methods will give a good correlation with ddPCR: if you make a dilution series, you'll find back a good correlation between both methods. However, the values might differ and that is perfectly normal!
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