What causes the smear below the band in electrophoresis?

A smear under the band in electrophoresis can be caused by multiple factors, including incomplete amplification, excess template DNA, too many cycles, inappropriate annealing temperature, and degradation of the DNA during the PCR reaction.

In your case, the PCR reaction has a short extension time of only 7 seconds, which could contribute to incomplete extension and result in smearing. Additionally, the number of cycles may be too high for the specific PCR reaction, leading to over-amplification and the production of nonspecific products. The annealing temperature could also be contributing to nonspecific amplification, as it is only 55.0°C, which may be too low for the specific primers being used.

To improve your results, you may consider optimizing the extension time and annealing temperature for your PCR reaction, reducing the number of cycles, and adjusting the amount of template DNA used. Additionally, you could try using a different DNA polymerase or optimizing the PCR conditions to reduce degradation of the DNA during the reaction.

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