You're probably getting two amplification products. Your primers might bind two sequences, or they might be binding each other. Easiest solution is to try new primers. Use the NCBI Nucleotide website (https://www.ncbi.nlm.nih.gov/nuccore) to find an accession number for your gene's mRNA sequence (begins with "NM_"), then use that as an input to design primers using NCBI's Primer BLAST tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Limit the product size to

It is possible to get a "double peak" from a single PCR product. This means that different regions of your PCR product have very different G/C content and different melting temperatures. Normally, if your PCR product is small enough (

sybr green and Evagreen chemistry normally works just like your PCR and these small molecules intercalates with the dsDNA during amplification with an increased fluorescence when bound to double strand DNA. Though it works effectively, one of the major problem with these chemistry is generates false positive signals; i.e., because the SYBR dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.

Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.

So, I request you to perform the melt curve analysis and as Katie & Tania suggested perform a PCR, run it on the Agarose gel and check for the non-specific amplification.

Also make sure your amplicon size should not be more than 250bp.

You can also go through this link for more information:

https://www.thermofisher.com/in/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/taqman-vs-sybr-chemistry-real-time-pcr.html

All the best!!