TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
7, 27	No bands were visible on gel – not even the DNA ladder	No nucleic acid gel stain was added	Soak gel in appropriate nucleic acid gel stain
	No PCR products were produced	Primers	Double-check primer design and, if required, resynthesize primers
		Too much template	Dilute template or boiled colonies an additional 5-fold
		Difficult template	Increase denaturation times; Use an alternate buffer or polymerase; Redesign primers
12	No SOE-PCR product	Error in primer design	Double-check primers, especially regions of reverse complementarity
19	A low number of colonies were recovered	Inhibited Clonase® reaction	Ensure that residual ethanol has evaporated before eluting SOE- PCR product
		Low transformation efficiency of E. coli cells	Prepare a fresh batch of competent cells; Buy competent cells from a preferred supplier
34	Cloned alleles have point mutations	Random errors, PCR “jackpot,” Bona fide SNP in genome of target strain	Use a high-fidelity polymerase for allele synthesis; Sequence additional clones; Repeat cloning process
	Cloned alleles have a point mutation in a region targeted by primer	Error in design or synthesis of primers	Check primer design; Resynthesize primers
36A or B	Cannot transform donor E coli cell line with allelic exchange vector	Restriction incompatibility with cloning strain used at Step 17	Use E. coli DH5α for cloning
	No merodiploids were isolated at selection.	Plasmid integration is low frequency	Repeat electroporation or biparental mating
		Use of incorrect selective media	Double-check antibiotics used in selective medium
		Target gene may be essential	No simple solution using the present protocol
	Lawn of bacteria on selective plates (i.e. selection escape)	Selective media was incorrectly prepared or degraded; recipient cell is antibiotic resistant; inoculum effect	Double check media preparation and antibiotic concentrations; choose a different antibiotic marker; dilute cultures an additional 10- to 100-fold before plating merodiploids on selective agar
37	Lawn of bacteria on NSLB + 15% sucrose agar (i.e. counter-selection escape)	Counter-selection escape; merodiploid has acquired inactivating mutation in sacB; allelic exchange vector has inactivating mutation in sacB; counter-selective media was incorrectly prepared	Repeat electroporation or conjugation with corrected merodiploid selection; repeat counter-selection; double check sequence of sacB in allelic exchange vector; double-check composition of counter-selective media
45, 47	Multiple bands on gel were produced by PCR reaction	Non-specific DNA amplification	Modify PCR protocol to increase its stringency; if multiple PCR products cannot be separated effectively by electrophoresis, use a nested sequencing primer for the mutant allele; redesign “Seq-F” and “Seq-R” primers
	No large insertion or deletion mutation was detected	Small sample size	Screen an additional 8 colonies isolated from NSLB + 15% sucrose agar
	Sequencing failed to identify a mutant with a SNP or short deletion or insertion mutation	Small sample size	Screen an additional 8 colonies isolated from NSLB + 15% sucrose agar
	No insertion or deletion mutation is detected after protocol is repeated multiple times	Target gene may be essential	No simple solution using the present protocol