Table 1.

Troubleshooting

Problem	Possible Cause	Solution
Sectored colonies. A single colony is composed of red and green cells.	Proper integration into the IIS-alphoidtetO-HAC occurred late during the colony’s growth and did not occur in all cells.	Cells with the correct marker can be rescued by dispersing the colony to single cells and sub-cloning.
	Concentration of counterselection agent insufficient to kill cells with incorrect marker.	Concentration of counterselection agent should be increased.
Cells are simultaneously red and green.	This can happen if ФC31 fails during the 1st round of integration (Figure 8b) or if ФBT1 fails during the 2nd round of integration (Figure 8d). In that case, a single cell expresses both PCF and GHT markers. Those cells can be eliminated only by Ganciclovir during the 1st round or by 5-Fluorocytosine during the 2nd round (Figures 8b and 8d).	Increase concentration of counterselection agent used, until two-coloured cells are dead. Then, restart experiment from the beginning, using this higher concentration of counterselection agent.
	Concentration of counterselection agent, Ganciclovir or 5-Fluorocytosine, is insufficient to kill the cells expressing the wrong marker.	Increase concentration of the appropriate selection agent.
Colourless (non-fluorescent) colonies.	Insufficient concentration of selection agent (Hygromycin or Puromycin) to prevent marker silencing due to hetrochromatin spread.	Slowly increase concentration of the appropriate selection agent in a stepwise manner. Check for reappearance of fluorescence as selection agent selects for cells with increased marker expression.
	Selection agents degraded.	If fluorescence does not reappear with increased concentration of selection agent AND the colony remained viable, check that the selection agent has not degraded from prolonged storage at 4°C or excessive heating from repeated freeze-thaw cycles. Discard old selection agent, and aliquot fresh stock of selection agents to vials of smaller volume before use. If selection agents are kept frozen, storage at −80°C is suggested.
Phenotype not matching expected genotype: ‘Fakes’	Heterochromatin silencing of one marker, giving the impression that one of the markers has been lost.	The colonies under investigation should be split, with one half preserved and other half for study.
		Screen out colonies that are ‘faking’ their genotype by using gradually increasing concentrations of selection agents to select for gene reactivation.
		Alternatively, apply HDAC inhibitor to (i.e., trichostatin a) to help reverse heterochromatin silencing. Be aware that the HDAC concentration used may kill the cells or/and cause HAC. destabilization.
Large numbers of ‘fake’ colonies.	Insufficient concentration of counterselection agent used.	Increase concentration of counterselection agent used.
	Too much time given for counterselection marker to degrade, allowing emergence of marker silencing by heterochromatin.	Decrease the time between transfection and application of counterselectable agent.
On application of selection agent, cells look unhealthy but do not detach from plate.	Lower than normal concentration of puromycin or hygromycin used.	Perform transformation in one well of a 6 well plate, then, 1 day after selection has begun, disperse cells to a 10 cm plate. Dying cells do not reattach.
		Increase concentration of selection agent, if possible.
Few colonies of the correct fluorescence obtained.	If carrier vector has a large genomic insert, there may be insufficient molecules being transfected into host cell.	Instead of co-transfecting the integrase plasmid and carrier vector, these transfections can be done sequentially. First transfect the host cells at lower than normal confluence with the integrase plasmid. Then, allow the cell culture to recover for a day before transfecting the same cells with the carrier vector.
		Instead of co-transfecting the integrase plasmid and carrier vector, these transfections can be done sequentially. First transfect the host cells at lower than normal confluence with the integrase plasmid. Then, allow the cell culture to recover for a day before transfecting the same cells with the carrier vector.
No colonies obtained.	HAC lost.	Maintain HAC selection with blasticidin.
	Selection agents.	Give sufficient time for building up of new selection marker and degradation of old counterselection marker. Both the timing and concentration are factors.
	Too much integrase expression vector used, leading to overexpression of integrase protein which form inactive protein aggregates. Too little carrier vectors used.	Restart experiment with empty vector and optimize quantity of integrase expression plasmid or carrier vector used before proceeding to actual experiment.
	Mutation in attBΦC31, attBΦBT1, or loxP sites.	DNA-sequence the platform cassette to make sure the integrase attachment sites have not mutated. Pick a different colony to use if mutations have occurred.
	Endotoxin contamination of DNA used in transfection.	Repurify vector with endotoxin free kit. Remember to use wash buffer and elute buffer that are both endotoxin free and filter sterilized. E. coli strains with reduce endotoxin may be used to produce the DNA.
	Standard 37°C incubation temperature of mammalian cell culture reduces activity of ΦC31 and ΦBT1 bacteriophage integrase	Lower post-transformation incubation temperature to 30°C for 2 days to promote recombinase activity before returning to normal temperature. Optimum temperature may vary with cell line.