I am trying to do a comparative experiment for differential expression of an unknown gene that is expressed in the brain. I got no Ct values for my unknown but got normal amplification and Ct values for my reference gene, which is why I am confused. Does anyone know why can this happen? if it was a pipetting error wouldn’t the reference gene give an error too?
I am attaching the amplification curve of my unknown gene.
Thanks
Hi, is it possible that the used primers can't anneal because of a different annealing temperature? It could help to check via a standard PCR with a temperature gradient and a following gel-electrophoresis. If you don't get a PCR product at all chosen temperatures, you may have to choose a different pair of primers. I hope, I could help you.
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