I have a qPCR-question. Maybe this is a trivial question. When we normally validated new primer, we make a dilution series from 1:10, 1:100, 1:1000…. Now our gene of interest is not highly expressed so the dilution series doesn’t work. So we want to make a dilution series for example 1:5, 1:25, 1:125…. The formula for efficiency calculation of the primers is (10^(-1/The Slope Value)-1)*100 But when we don’t use 1:10, 1:100… did we then have to change the 10 in the formula for example to 5?
Hi Sven,
The slope of the line is the same whether you use 1:10, 1:100, 1:1000 vs 1:5, 1:25, 1:125. Your data points are just closer together with smaller dilution steps.
Hi Sven,
The slope of the line is the same whether you use 1:10, 1:100, 1:1000 vs 1:5, 1:25, 1:125. Your data points are just closer together with smaller dilution steps.
Also, you do not need to use your actual cDNA for this: for primer amplification efficiency, just using known quantities/dilutions of a PCR product is sufficient.
After all, even if you start with cDNA, ~99.999999% of the amplicons the reaction will be replicating will be PCR products themselves.
I've used 1:2 dilutions for low-expression genes before with no problem. As Katie A Burnette says, the slope of the line should be the same regardless of dilution factor used.
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