Hello, I have been running qPCR (on Qiagen rotor-gene machine) to amplify one of the RHD gene exons with perfect melting curve peak. A sudden weird melting curve peak appeared with last runs. To troubleshoot, I changed the Sybergreen MasterMix (same lot no), used filtered tips and made new primers from the stock I had without any good results. I have attached RHD positive control peak from previous run (good melting curve peak) and new run (weird melting curve peak). Please note that I am using the same protocol for each run.
Could you please share your experience what could be the reason or any additional tips to troubleshoot?
It looks contaminated with a fair number of non-specific primer/template interactions. Have you run the product out on a gel (2% agarose aught work fine) to check for multiplte/single bands?
Thank you Mathew for sharing your experience,
In this case, I might need to prepare new RHD+ DNA sample (as the sample might be contaminated). I am planning to run the gel tomorrow,
The weird overlapping peaks look like they might be primer dimers as they appeared before 79oC and desired peak should be at 81.5oC (as per RhD+ control in previous successful runs)
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