Standard curve dillutions not 3 cycles apart - (Oct/12/2006 )
I have run two standard curves (polio virus) on a LightCycler using a 10-fold dillution series but my CP values are not 3.3 cycles apart. I am getting almost 6 cycles between my 1 and 1:10 dillution. I am hopeing it's not operator error as these are easy dillutions. Could it be the Qiagen RNeasy kit? Could it be something else?
No offense meant, but this sounds like a classical pippetting error - samples containing 1/10th of each other have to show up in 3-4 (doesn't need to be 3.3 exactly as this is the theoretical value (log210=3.33) cycles distance to each other on RT-PCR. Try to redo your experiment and watch out if you dillute your sample unintentionally more than you should....
I'm not offended. I would prefer it to be a pippetting error because I could fix that. I am worried because the person who was training me gave me different instructions for the standard curve then what he used in his last two published papers, even though we are performing virtually the same experiment. He used sonicated herring sperm as carrier DNA; I have never heard of using heriring sperm until I re-read his papers. Could not using herring sperm be my problem?