Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Library yield is significantly lower than expected based on input amount, despite successful completion of all enzymatic steps.
Significantly lower library yield after SPRI bead cleanup steps, with visible reduction in final library concentration disproportionate to expected recovery rate.
Adaptor or primer peaks visible on Bioanalyzer or similar instrument after PCR amplification, indicating inefficient cleanup or excess reagent usage.
High molecular weight fragments appear on Bioanalyzer after PCR due to single-stranded library fragments and heteroduplex formation when PCR primers are depleted. Data quality may be compromised upon sequencing.
No library visible on Bioanalyzer or similar instrument after amplification, or library fragments remain the same size as input DNA instead of showing expected ~120 bp increase in size.
Sharp 127 bp peak visible on Bioanalyzer representing adaptor dimers, reducing the proportion of desired library fragments and overall usable yield.
Library fragment size distribution is shifted from expected range, with either smaller or larger inserts than intended, or discrepancy between fragment analyzer measurement and sequencer-determined insert size.
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