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Standard Curve calculation

Standard Curve calculation - Real-Time PCR and Quantitative PCR Forum

Standard Curve calculation - Real-Time PCR and Quantitative PCR Forum


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Old 10-25-2010, 07:29 PM
Pipette Filler
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Default Standard Curve calculation



I'm working with a Roche Lightcycler 1.5 and want to do relative quantification of three genes. I just started to create a reference standard curve of my housekeeping gene. I used 5 point serial dilutions of cDNA and when checking my results I had an efficiency of 3.4. I know this is theoretically impossible, the curious thing is every time I take off a dilution point from the curve, the efficiency comes closer to 2. What did I do wrong?
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Old 02-10-2011, 04:47 AM
Pipette Filler
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Default Re: Standard Curve calculation

Do you have contamination? Usually your low concentrations will start to have Ct values that level off if contaminated, therefore resulting in a less negative slope (inflated efficiency). Does your NTC come up positive?
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