Nonspecific Amplification Products (PCR / RT-PCR Amplification Problems)

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Extra, unwanted bands appear on gel in addition to or instead of the target product, indicating off-target amplification. Common causes: 1) Primers hybridizing to secondary sites on template DNA 2) DNA contamination introduced in primers or reaction buffers 3) For RNA templates: genomic DNA contamination in RNA preparation 4) Excessive template concentration enabling nonspecific priming 5) Primer concentration too high promoting off-target binding 6) Thermal cycler temperature inaccuracy Solutions: 1) Redesign primers with reduced specificity for secondary sites; increase annealing temperature by 2°C to 5°C increments 2) Run negative control (no template); prepare fresh materials if contamination detected 3) For RNA: Perform DNase treatment and completely inactivate/remove DNase, or re-prepare RNA using different method 4) Titrate template amount to optimal concentration for your system 5) Titrate primer concentration to optimal level for your system 6) Repair/calibrate thermal cycler or use different machine