The primers were poorly designed.

Dr Demetriou explains that considering that the positive control elicited the correct band, it is unlikely that the problem is with any of the reagents that are shared between the control and sample preparations. He goes to explain that it is true that poor primer design can lead to primers that are not specific to the target sequence, have secondary structures, or form primer-dimers that can result in inefficient or non-specific amplification. However, your supervisor explains that the primers have been successfully used many times in the past so poor design of primers is not the most likely explanation. Dr. Demetriou asks you to reconsider the most likely reason for these results.