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Overcycling occurs when too many PCR cycles are used for the final library amplification. In this case, when the PCR reaction runs out of primers and template and generated ds-cDNA starts to denature and reanneal improperly. This results in longer, bulky molecules that migrate at a lower speed on the Bioanalyzer chip or gels. This can interfere with exact library quantification for pooling and loading for sequencing if relying solely on the Bioanalyzer results.
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