qPCR amplification of non template controls

[bsengez]

Hi,
We’re using SYBR green qPCR master mix from Fermentas to check the expression level of our transfected cell lines. I’m getting a signal in my template free negative control (nuclease free water instead of template) after 23th cycle. I also confirmed it on gel. I have changed supply of nuclease free water and purchased new primers and still get a signal. I couldn’t find what is wrong.

[Mikael Kubista]

Most likely you have contamination. Make sure to replace all your reaction mixes. If it does not help consider redesigning your assay. TATAA Biocenter (http://www.tataa.com) offers help with that, if you are interested. Consider also using UNG.

[sylvia.c.wong]

Did you check your melting curve? If there are two peaks in your no template control but one of the peak disappears in your "undiluted" sample then the signal is mostly from primer dimers.