Hello

It is the pipetting error, since the standards had the same types of template and other ingredients.

For the one sample, the peak is shifted by 1.0 C:

Most were at 80.5C, the unusual peak was at 79.5C

I ran the products on a 2% agarose gel and all the samples had 1 crisp band at the right size -- including the sample with the unusual melt curve.

I didn't see any signs of primer dimers and I don't think I simply ran them off the gel as the 75 bp band from the ladder was only halfway across the gel.

I'm inclined to believe the amplicon is correct and the 1C difference is just measurement error, however, the Ct for the sample was a bit higher than was expected of the sample.

Pipetting error is possible, but they were all made with the same master-mix. The only thing that was added individually was the cDNA. I did not omit the cDNA for that sample as the volume was as expected. The cDNA stock for the unusual sample was diluted for use in other samples -- which were fine -- so I doubt it is the problem.

Sometimes you can have secondary structure in the right sample that in effect creates the wrong peak. Why this sample and no other?....It happens

If you have verified the band on an agarose gel don't worry