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A second peak between 1,000 – 9,000 bp, or an elevated baseline on a bioanalyzer trace that extends underneath the upper marker is an indication of overcycling (Figure 41). Similar features can be seen with other types of microcapillary electrophoresis traces.
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Figure 1 | Correct versus overcycled QuantSeq Library Results: Correctly cycled (10ng UHRR, 19 cycles, blue), overcycled (10ng UHRR, 24 cycles, red). Low input protocol modifications were used (skipping step 2, 1 hour incubation at 42 degrees C at step 4, reduced volumes of PB and PS at steps 16 and 29).
What causes overcycling?
Overcycling occurs when too many PCR cycles are used for the final library amplification, when the PCR 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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