Table 3 |.

Troubleshooting table.

Step	Problem	Possible reason	Solution
5	Poor or no amplification.	Ill defined	Due to low complexity, the QCgRNA PCR is robust and has so far never failed to generate full-length product. Yields, however, may vary slightly, possibly due to QCgX primer differences. If yields are very low, purify and concentrate the QCgRNA amplicons prior to transfection.
16	Multiple bands or smear.	Non-specific priming; non-optimal template concentration.	Include 5% DMSO in the PCR; slightly increase the annealing temperature and/or titrate MgCl2 (test 0.1–2.5 mM final concentration in the PCR); titrate template concentration; in rare cases, re-design primers.
	No or very little PCR product	Amplified region is particularly GC-rich.	Add 5% DMSO and/or 0.2 M betaine to the PCR; increase the concentration MgCl2in the PCR and/or lower the annealing temperature; Exchange TEMPase DNA polymerase with KOD DNA polymerase; in rare cases, re-design primers so as to amplify a less GC-rich region around the target site.
21	Signal intensity is too high/saturated.	Concentration of IDAA PCR sample is too high;	Decrease sample concentration by diluting PCR products further in Step 17 before addition of the formamide/size standard mix;
	No signal or too low signal intensity.	Too little sample injected.	Increase sample concentration by diluting sample less in Step 15.
		Degraded or incorrectly stored formamide.	Use fresh formamide.
		Air bubble at the bottom of sample well.	Centrifuge the sample plate briefly before inserting it into the fragment analyzer.
	Two peaks in control (i.e. wt) samples: a prominent wt peak and a minor −1 bp peak.	Incomplete 3´ A nucleotide addition to the IDAA amplicon by the polymerase.	Increase the final extension step in cycle 41 to 60 min (Step 20).
			In the rare cases, when the problem persists, add a single G nucleotide over-hang85 or a GTTTCTT “PIG tail”86 to the 5´ end of the IDAA Rev primer, which generally will promote complete 3´ A nucleotide addition.