I am quite new to real time PCR, so maybe I don't see my fault. I isolated RNA of bacterial cell culture of stationary phase and transcripted it to cDNA. The RNA looks fine on the gel. When I do qPCR using SYBR green I get very distinct CT for the replicates. Before (log-phase RNA) it worked fine. I also have now suddenly low CT in my non-template control, although I took everything (incl. water) freshly. Recently our freezer broke down and temperature rose to -7 °C overnight. Could the primers maybe just have been degraded? Everything else was replaced.
Hi thank you. I do technical replicates (#2) and to exclude pipetting error I first make a master mix and later just add the cDNA. Previously the primers worked fine for log-phase cDNA. I am just wondering why I have such low Cts in my non-template control. Maybe I just took an old SYBR green aliquot?
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