Dear All,
I used HepG2 as positive control as my GOI is confirmed to be expressed in HepG2. It had a melt peak. But there was one minor peak adjacent to 81C peak. May I know what is the problem? Primer Dimer? Both were pipetted out from same mastermix tube.
Your minor peak at your sample amplification most probably corresponds to an unspecific binding that could be directly related with the template quality. Since, in your control reaction, you did not observe a minor peak close to the major peak of 81ºC, that might indicate primer dimer. I suggest to improve your templete quality extraction and or synthesis (if you were using cDNA).
Try running your amplicon through https://www.dna.utah.edu/umelt/umelt.html
which predicts your melt curve. Repeat your PCR run with diluted DNA; if the problem persists, repeat extraction
Dear Stephen,
I have run through UMelt. The estimated melting temperature will be 83C but my actual Tm is around 80.5C.
Hello Shin Wei,
It seems you have an unspecific amplicon which melts at a lower temperature using these primers. You can verify if you have additional bands by running your final real-time RTPCR reactions on a standard agarose gel with EtBr. As Aaron suggested, you can try to improve your template quality (e.g. DNase treat if it's a genomic amplification) or redesign primers.
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