FAQs

A high background level of fluorescence is most likely due to an excess of template in the reaction. This relates to the qPCR instrument picking up the SyGreen dye which binds to the template DNA. Samples usually contain a lot of DNA other than the target gene, so there will be enough present to register fluorescence. We recommend diluting the samples 100x-1000x to overcome this issue.

This should not only reduce the background signal, but also allow an accurate quantification because the dilution should bring the Ct values in the range of those obtained with the standards. Keep in mind that if the Ct is earlier than the first standard, it won’t be considered accurate enough quantification for publication.