Common issues & fixes
| Symptom | Likely cause | Fix |
|---|---|---|
| Low yield | Bead ratio too low, or beads not fully resuspended | Use 1.8× (higher for low input); vortex the bead stock to homogeneity before dosing. |
| RNA lost / pulled off | Aspirated while the supernatant was still cloudy | Let the magnet settle until clear before aspirating — vision gates this step. |
| Poor wash / salt carryover | Ethanol not fresh or wrong percentage | Make 70% ethanol fresh that day; >70% washes poorly, <70% strips RNA. |
| Hard to resuspend / low elution | Beads over-dried | Dry just until no visible ethanol, not cracked; mix fully during elution. |
| Ethanol carryover into next step | Beads under-dried | Extend the dry slightly and ensure full ethanol aspiration before eluting. |
| Degraded product / fails integrity QC | RNase contamination or warm RNA dwell | Use nuclease-free consumables; keep RNA cold after elution. |
When not to automate this
For a few samples occasionally, manual SPRI may be faster than deck setup. If you must minimise immunogenic double-stranded RNA, SPRI alone won't fully remove it — pair it with an orthogonal polish (oligo-dT capture if your transcript is poly(A)-tailed, or off-bench cellulose/HPLC). Confirm the exact bead ratio and per-well volumes against your validated SOP before production use.
Verification: this protocol is reviewed — simulation-verified and scientist-checked, but not yet vision-verified on hardware. Validate against your controls on first run.