i am doing primer efficiency test, and i do 1:10 dilution for cdna
some efficiency was more than 100, like 178%, however, some efficiency was like 50000000, i never see the efficiency shows 100%, where is wrong? thanks
Hi Xiaoyu Qi,
Firstly, your threshold looks quite low and may be causing some issues, but there is definitely other problems here. You are testing a 1:10 dilution series so, assuming perfect pipetting and perfect efficiency, your curves should be 3.32 cycles apart (this is shown on your software as 'slope'). Not only is this not the case for some, but more worryingly you have some low copy samples as being more positive than high copy samples - hopefully this is also due to the low threshold setting but if not it suggests either incorrect pipetting/labelling or a sub-optimal assay.
I'd start start by reading the manual and seeing if you can correct some of the issues with the information in there, if that doesn't work you need to investigate both operator error (it happens to all of us) or assay suitability.
Hope that helps
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