I've been having a rather odd problem with my standard curve. The efficiency is great, usually 90-95% and my R^2 values are near 0.99. Although this sounds odd, Ct values seem too high. My curve ranges from 1e7-1e1 copies. I seem to be detecting 10 copies of my standard at a Ct of about 31. That just doesn't seem right 10 copies is a very low amount. I'd except my Ct values in that range to be much lower. I know that you can't necessary compare Ct values between different runs but something just seems off.
I first thought that I performed my calculations of copy number wrong or I messed up my dilutions. I had my calculations tripled checked by others and I prepared my standard curve multiple times.
Next I thought that I incorrectly quantified my standard. Perhaps I have more concentrated DNA than I thought? I measured the plasmid for my standard curve with a nanodrop but perhaps somehow the quantification is not accurate? I've triple checked the quantification. My plasmid is linearized. I avoid multiple freeze thaw cycles of my standard and have it aliquoted.
I don't think it's a primer or probe issue because my primer probe set has been successfully used by other labs and published in several papers.
Can I have someone's opinion?
Also, I have an odd contamination problem in my no template control. I get a Ct values of about 36 in my NTC but in my negative control samples (serum samples of uninfected mice) I always get undetermined. I am very careful with my water. It's aliquoted and the bottle is only opened in the tissue culture hood so I don't know where my contamination issues are coming from? Also I would expect to see contamination in my negative control if I also see it in my NTC.