What should I do if the cDNA-enriched product fails QC? (Concentration < 0.5 ng/μL, or the main peak is not within 750–2500 bp.)

Possible causes:

• Insufficient input cells, inaccurate counting (live cells loaded < 1,000, below the protocol minimum).
• Improper use of DNA selection magnetic beads (insufficient mixing or incorrect bead:product volume ratio — not 0.6×).
• Beads over-dried during ethanol step (room-temperature drying > 1.5 min), causing bead cracking and poor elution.

Recommended Solution:

• Correct cell input and bead ratio: count cells and viability with a cell counter (e.g., Countstar IN030101 or SeekGene M002C); ensure viability > 90% and input ≥1,000 and ≤24,000 live cells. Vortex DNA-selection beads 30 s before use and add beads at 0.6× (e.g., 30 μL beads to 50 μL PCR product).
• Beads drying and elution procedure: after 80% ethanol washes, air-dry beads for ~ 1 min (beads should be matte and not cracked), then immediately add 24 μL nuclease-free water. Vortex 10 – 15 s, quick-spin, pipette 15× to resuspend, incubate 2 min at room temperature, place on magnet, then transfer 23 μL supernatant to a new tube to ensure full elution.
• Low-yield rescue: if input cell number was low (e.g., ~12,000) and pre-cleanup enrichment product concentration is < 10 ng/μL, return to the thermocycler and add 1 – 2 additional PCR cycles before purification. If > 10 ng/μL, proceed to purification.
• Evaluation decision: if cDNA = 0.5 – 1 ng/μL (borderline) and main peak 750–2500 bp → you may proceed to library prep with caution; if cDNA < 0.5 ng/μL or peak is off-range → do not proceed to library prep.