Common issues & fixes
| Symptom | Likely cause | Fix |
|---|---|---|
| No/few colonies | Poor annealing, ligation, or transformation | Verify the annealing program, ligase activity, and competent-cell handling; run a positive control. |
| High background (empty vector) | Incomplete vector digestion | Confirm the backbone is fully digested/dephosphorylated before the run. |
| Wrong insert on screening | Oligo design or orientation error | Recheck oligo design and overhangs; sequence-verify positives. |
| Run paused mid-protocol | Vision flagged a plate/tip exception | Re-seat the flagged item as prompted; the robot resumes from that step. |
When not to automate this
This protocol is currently simulated — validate cloning efficiency against your manual baseline before scaling. For large guide libraries, pooled synthesis/cloning approaches may be more efficient than arrayed assembly.
Verification: this protocol is simulation-verified — confirmed to run in the simulator but not yet run on hardware by RoR. Validate before using with critical samples.