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qPCR primer efficiancy - (Aug/17/2007 )

When calculating efficiency, I make a standard curve in 10 fold dilutions, do each dilution in the series in triplicates. Take the average ct value and plot this over the diltion factor and use the slope to calculate efficiency. Okay so I've done this. My ct's are close to 3.3 apart between dilutions but I'm getting a weird number for my slope. Am I doing this wrong? Do I need a log scale for the DNA dilution factor? My slope is like -5. It should be way closer to 2. My average ct values for 10 fold dilutions are 13.2, 16.4, 19.6. What am I doing wrong?

-sumogirlie-

QUOTE (sumogirlie @ Aug 18 2007, 04:47 AM)
When calculating efficiency, I make a standard curve in 10 fold dilutions, do each dilution in the series in triplicates. Take the average ct value and plot this over the diltion factor and use the slope to calculate efficiency. Okay so I've done this. My ct's are close to 3.3 apart between dilutions but I'm getting a weird number for my slope. Am I doing this wrong? Do I need a log scale for the DNA dilution factor? My slope is like -5. It should be way closer to 2. My average ct values for 10 fold dilutions are 13.2, 16.4, 19.6. What am I doing wrong?



Hi,

You need to convert your dilution concentrations to log values, so you would end up with something like the following (3,13.2),(2,16.4),(1,19.6) which will give a slope of -3.2. Your efficiency is then 10^(-1/slope), giving ~2.05.

Hope that helps.
Cheers,
Anil.

-maset-

Try to find ABI PRism User Bulletin #2. It is explained very well in there. Or try using a web based analysis system such as miner, which can calculate the efficiency from the raw values of your amplification curves (so you don't need to always do serial dilutions).

-kr├╝melmonster-