Why do I get an unexpected band or multiple bands for my PCR result?

The unexpected or multiple bands that you are experiencing in your PCR results, is most likely the result of nonspecific binding or the formation of a primer-dimer. Try the following tips to remedy this issue:

• Avoid excessive cycling as this can increase the chance of nonspecific amplification, for most PCR applications 25-35 cycles is sufficient
• Avoid impurities and contaminates in your PCR components as these can inhibit PCR, and can lead to incorrect amplification
• Avoid long extension times, for most PCR applications an extension time of 1 min/kb is sufficient
• Avoid long annealing times as this can cause spurious priming, for most PCR applications the typical annealing times are 15 to 30 seconds
• Optimize annealing temperature, at low temperatures primers can bind nonspecifically to the template
• Check primer design