Standard Curve calculation

[Alejandro Iregui]

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?

[thehulk]

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?