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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 (see Figure 1). 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).
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