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 Concentration of counterselection agent insufficient to kill cells with incorrect marker	Cells with the correct marker can be rescued by dispersing the colony to single cells and sub‐cloning Concentration of counterselection agent should be increased
Cells are simultaneously red and green	This can happen if ФC31 fails during the first round of integration (Fig. 8B) or if ФBT1 fails during the second round of integration (Fig. 8D). In that case, a single cell expresses both PCF and GHT markers. Those cells can be eliminated only by ganciclovir during the first round or by 5‐fluorocytosine during the second round (Figs. 8B and 8D). Concentration of counterselection agent, ganciclovir or 5‐fluorocytosine, is insufficient to kill the cells expressing the wrong marker	Increase concentration of counterselection agent used until two‐colored cells are dead. Then, restart experiment from the beginning, using this higher concentration of counterselection agent. Increase concentration of the appropriate selection agent
Colorless (non‐fluorescent) colonies	Insufficient concentration of selection agent (hygromycin or puromycin) to prevent marker silencing due to heterochromatin spread Selection agents degraded	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. 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 histone deacetylase (HDAC) inhibitor to (i.e., trichostatin a) to help reverse heterochromatin silencing. Be aware that the HDAC concentration used may kill the cells and/or cause HAC destabilization.
Large numbers of “fake” colonies	Insufficient concentration of counterselection agent used Too much time given for counterselection marker to degrade, allowing emergence of marker silencing by heterochromatin	Increase concentration of counterselection agent used 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	Make sure all plasmid and BAC used are free of bacterial genomic DNA. Use exonuclease to remove linearized DNA. 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 Selection agents Too much integrase expression vector used, leading to overexpression of integrase protein which form inactive protein aggregates. Too little carrier vectors used. Mutation in attBΦC31, attBΦBT1, or loxP sites Endotoxin contamination of DNA used in transfection Standard 37°C incubation temperature of mammalian cell culture reduces activity of ΦC31 and ΦBT1 bacteriophage integrase	Maintain HAC selection with blasticidin Give sufficient time for building up of new selection marker and degradation of old counterselection marker. Both the timing and concentration are factors. Restart experiment with empty vector and optimize quantity of integrase expression plasmid or carrier vector used before proceeding to actual experiment 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. 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 reduced endotoxin may be used to produce the DNA. 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.