Table 1.

Problem	Possible cause	Solution
No transformants	Low transformation rate	Repeat protocol with as many replicates as manageable.
	Degraded anhydrotetracycline	Prepare fresh stocks of anhydrotetracycline. Avoid freeze-thaw cycles.
	Poor quality of deletion construct plasmid DNA	Confirm plasmid size and quality by separation through agarose gel. Extract plasmid from larger volumes of liquid culture of E. coli for cleaner, more-concentrated plasmid DNA.
	Deletion construct contains errors in plasmid genes essential for chlamydial maintenance	Sequence the deletion construct. Re-clone if necessary.
	Over-passaged McCoy cells hindering rate of chlamydial infection	Use low-passage number McCoy cells.
	Recovery time insufficient for propagation of the particular deletion construct	Extend the recovery time of BASIC PROTOCOL 2, step 5 from 7 hours to under 12 hours.
Deletion construct is not depleted from the chlamydial population upon removal of anhydrotetracycline from medium	Deletion construct contains sequence errors interfering with expression of tetR or repression of pgp6	As a technical positive control, transform with empty pSUmC, and ensure regulation of plasmid stability is functioning properly. Sequence the deletion construct for errors. Re-clone if necessary.
No exclusively green-fluorescent inclusions develop after multiple rounds of re-infection of transformants in the absence of anhydrotetracycline	Homologous recombination arms of deletion construct do not match chlamydial genomic sequence	Amplify and sequence arms from chlamydial genome and deletion construct, and ensure sequences match exactly. Re-clone if necessary.
	Target gene may be essential or deletion may cause severe growth defect	Clone a deletion construct targeting a non-coding, intergenic region of the chlamydial genome as a technical positive control. If the positive control functions properly in C. trachomatis, propagate the transformants carrying the desired deletion construct under varying conditions, ie, different host cells, concentrations of selective pressures, and times between rounds of harvesting progeny and re-infecting new monolayers, in order to provide the required growth conditions for propagation of the chlamydial mutant.