What is the most likely reason for an unexpected band in a PCR reaction?

The most likely reason for an unexpected band in a PCR reaction relates to the annealing temperature being too low. The annealing step in PCR is crucial because it allows the primers to bind specifically to the target DNA sequence. If the temperature is set too low, it reduces the specificity of primer binding, which can result in non-specific amplification or the binding of primers to unintended sequences. This can lead to the formation of unexpected bands in the resulting gel electrophoresis, as the primers may amplify sequences that were not the intended targets.

In contrast, using primers that are too short can lead to issues such as reduced specificity, but it is the low annealing temperature that directly increases the likelihood of non-specific amplification. Repeating the PCR too many times might influence the overall yield and quality of the amplification but is less likely the direct cause of unexpected bands than inappropriate temperature settings. Not using a control sample is important for validating the results but does not directly cause unexpected bands in the reaction itself; it merely complicates the interpretation of the results. Thus, the primary factor contributing to the unexpected outcomes in this scenario is the annealing temperature being too low.