Plasmid standard problem - (Dec/01/2010 )
I used plasmid as standards. After serial dilution ,ie. ranging from 10E5 to 10E0, my qPCR result often showed that the Ct value of low concentration template was less than that of the template with high concentration. Indeed , the dilution process was carefully adjusted. The contamination of the reagent or primers were sorted out. Now I don't know how to get rid of this problem.Any one could give some advice?Thanks.
Typically when your low concentrations show lower than expected Ct values it is because the assay is simply not robust enough to detect such low concentrations of template. One easy solution is to use a standard curve with higher concentrations of template (for example from 10E2 to 10E7). Of course if you need to detect such low levels of DNA (10E0) then you may have to re-design your assay.
Thanks for your suggestion. My result not performed well for the serial dilution, such as Ct=25 using 10E3 template, while Ct=24 using 10E1 template, etc. I don't know why the low conc template gave the positive signal more quickly than the relatively high conc template.