What should I do if the cDNA-enriched product contains small-fragment contamination?(Agilent/Qsep shows fragments < 250 bp.)

Possible causes:

• 0.6× bead selection may not capture small fragments efficiently— small fragments remain in the supernatant.
• Incomplete 80% ethanol washes (ethanol not freshly prepared or insufficient washes), leaving salts and reagents that interfere with size selection.

Recommended solution:

• Secondary purification to remove small fragments: add DNA-selection beads again at 0.6× (e.g., for 50 μL product add 30 μL beads). Pipette 10× to mix, incubate 5 min at room temperature, quick-spin, place on magnet until clear, remove supernatant, then repeat the ethanol washes and elution per Step 2–3 until small fragments are removed.
• Ethanol wash best practices (preventive): prepare fresh 80% ethanol (use within 24 hrs). For each wash add 300 μL, incubate 30s at room temperature, then carefully remove supernatant. Perform two washes. After the final wash, quick-spin, remove residual ethanol completely using a 10 μL pipette tip to avoid salt or ion carryover (Mg²⁺, Na⁺) that can affect downstream steps.