It would help to know a little more information, such as the band sizes and extension time of your PCR. If the upper band is larger than reasonably expected from the PCR conditions (general guideline is 1 min per KB). So, if your extension time is 1 min and the band is much larger than 1 KB it's probably something from your template, such as a template (plasmid/gDNA). Is the far right land your negative control? There is no upper band so that would be consistent with a template source for the band. What if you just load some template on your gel without running PCR? Does that show the upper band? Hope this helps!

The larger band is very strong and much bigger than the correct one. One possibility is that it is formed from a repeat sequence. I would try a30 second extension time and 10 second annealing time on your pcr. Also use much less dna template, It would also be interesting to run 2 samples one with only forward primer and one with reverse primer only to see if one primer can generate the band which can happen with inverted repeat sequences.

Run your primer sequences on primerblast to look for multiple hits on yout genome

Are you trying to amplify your genes from genomic DNA? if so, how much 'ng' of template DNA did you add in the PCR reaction mix? Try running the same amount in empty wells parallely. Maybe, it is just saturation of your template DNA.

Thanks Amy Klocko for your input.

232, 390, 271, 621, 798 and 232; extension time is 50s at 72°C for 36 cycles.

Yes, the band is larger than 1kb size

Thanks

If the band keeps appearing in all reaction this might be contamination (in MMX, primers or DNA template)

If not

I would try diluting the components (e.g 1:80) of the reaction (DNA template and primers) interchangeably, this might help you narrow down the culprit

All the best

My guess is that is DNA template. It's much larger than expected from a 50 second extension time. How much template are you adding? Seems worth quantifying your template and reducing the amount of template per reaction.

Dear Ezeador,

As Amy Klocko and Papri Basak pointed out it is actually your template DNA. if you see the ladder position and the PCR band its way beyond your expected PCR product size (you can try reducing the template concentration you may get clean gel).