I have attached an amplification plot from a qPCR assay I am trying to optimise. The efficiency of the reaction is fine (99%) however the curve just does not have a nice sigmoid shape. The standard curve is generated using plasmid DNA however the assay fails to amplify positive control genomic DNA.
It has been suggested to me that I need to perform a temperature titration as the annealing temperature may be too high. As far as I know the qPCR machine that I use does not have the ability to perform thermal gradients. Knowing this is it worthwhile for me to perform the temperature titration using the same primers in a conventional PCR assay?
Any ideas/tips/feedback on what you may think is going on with this assay is greatly appreciated.