TABLE 3.
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 |