What should I do if the cDNA fragment-size distribution is abnormal? (Agilent 4200 or Qsep shows no 250–5000 bp fragments, or the main peak is outside 250–2500 bp)

Possible causes:

• Amplification mix was prepared incorrectly (e.g., insufficient small-volume cDNA Primers — 2 μL missing — or primers not fully mixed with the 2× PCR Master Mix).
• PCR program settings are incorrect (e.g., lid temperature not set to 105 °C causing evaporation; incorrect cycle number).
• Poor cell quality — especially nuclei preps where nuclear integrity is compromised.
• Incomplete mixing of Cleanup Mix with the aqueous phase during purification, causing loss of cDNA in the supernatant.

Recommended Solution:

• When preparing the amplification mix, add large-volume reagents first (2× PCR Master Mix), then pipette small volumes (use a suitable 2.5 μL pipette for primers). Add purified cDNA last and mix thoroughly.
• Verify and correct the PCR program before running (confirm lid temperature = 105 °C and the recommended cycle number).
• Only proceed with samples that meet input quality standards (replace or reprocess poor-quality samples).
• During cDNA cleanup: after adding Cleanup Mix, pipette gently and slowly ≥15 times (keep the tip at the liquid surface to avoid splashing), incubate at room temperature for 10 min, and pipette gently 10 times again at every 5min to ensure full bead–cDNA binding. During magnetic capture, aspirate from the side opposite the beads 5× to accelerate clearing before removing supernatant — this helps prevent cDNA loss.