Table 6. Troubleshooting table.

Step(s)	Problem	Possible Reason	Solution
6	No ZFN target sites identified	Insufficient length of sequence to identify an OPEN target site	Consider expanding the DNA search length
6	Too many ZFN target sites identified		Consider using only sites that contain two or more GNN subsites
16, 58	No transformants obtained from ligation	Low efficiency of transformation	Consider re-making competent cells or purchasing commercially available competent cells.
16, 58	No transformants obtained from ligation	Insufficient concentration of vector backbone or insert	Check DNA concentrations using 260 nm absorbance readings on a spectrophotometer.
16, 58	Too many transformants from vector backbone control ligation	Incomplete digestion of vector backbone	Screen additional colonies from the actual ligation/transformation plate or consider re-digesting vector using higher concentration of enzyme or less DNA
26	Number of colonies on TK and TKS plates are the same	sacB gene in the reporter plasmid acquired a mutation	Repeat steps 20-26 using another plasmid candidate
36	Failure to obtain transformants	Low efficiency of transformation	Consider re-making competent cells (step 35)
36	Failure to obtain transformants	Insufficient amount of pAC-alphaGal4 plasmid used for transformation	Use larger amount of DNA for transformation and/or plate a larger portion of the transformation
43	Failure to obtain PCR product on gel	PCR reaction was unsuccessful	Verify that you are using Expand PCR enzyme and that PCR cycling conditions are correct
43	Failure to obtain PCR product on gel	Low amounts of DNA template in PCR reaction	Verify that the pools (obtained from the Joung lab) were fully resuspended by checking DNA concentration on a spectrophotometer.
65	Number of transformants from pre-amplification quantification is too low	Low efficiency of transformation	Consider re-making electrocompetent cells or purchasing commercially available electrocompetent XL1-blue cells.
65	Number of transformants from the positive control is adequate but number of library transformants is too low	Insufficient quantity of library DNA	Repeat step 60 with remaining half of ligation and repeat electroporation. If this fails, try repeating the ligation.
71	Titers show that <50% of cells were infected by helper phage	Titer of helper phage was too low to successfully superinfect the culture	Repeat steps 66-71 using a larger volume of helper phage or consider re-making high-titer helper phage stock.
74	Failure of selection strain culture to grow to correct density	Colonies on plate were older than 1 week	Re-streak strain and repeat innoculation with fresh colonies
74	Failure of selection strain culture to grow to correct density	Glassware used for innoculation contained residual soap detergent	Thoroughly rinse glassware with distilled water before autoclaving to remove any residual detergent.
74	Failure of selection strain culture to grow to correct density	Insufficient colony dispersal during innoculation	Repeat innoculation making sure to completely disperse colony in media
74	Failure of selection strain culture to grow to correct density	Incorrect composition of NM media	Verify that M9 salt stock is actually 10× (not 5× as described by recipe on the bottle supplied by Difco)
74	Failure of selection strain culture to grow to correct density	Incorrect composition of NM media	Verify that glucose used was obtained from Mallinckrodt-Baker
74	Failure of selection strain culture to grow to correct density	Incorrect composition of NM media	Verify composition of all other NM reagents
74	Failure of selection strain culture to grow to correct density	Inability of strain to grow in NM media	Go back to the “B” candidate from step 35, repeat step 36 and proceed with selections using this strain.
80	Total number of cells in 250 μl of resuspended cells from step 77 is not at least three-fold more than the total number of infected cells.	Too much phage library was used for the selection	Repeat steps 72-80 using an appropriately decreased number of CTUs (carbenicillin-transducing units)
80	The total number of infected cells in 250 μl of resuspended cells from step 77 is <2.5 × 106	Too little phage library was used for the selection	Repeat steps 72-80 using an appropriately increased number of CTUs (carbenicillin-transducing units)
90	Titer of the enriched zinc finger library phage stock is not >1×104 ATU/μl	Too little phage library was used for the selection	Concentrate the enriched zinc finger library phage stock and re-titer the concentrated stock as described in Box 8.
97	The total number of cells in the culture of step 94 is less than three-fold the number of infected cells	Too much enriched zinc finger library phage was used for infection	Check calculation of enriched zinc finger library phage titer. Consider repeating steps 85-90 to obtain an accurate titer.
97	The total number of infected cells plated on the gradient plate is <2.5 × 106	Too little enriched zinc finger library phage was used for infection	Check calculation of enriched zinc finger library phage titer. Consider repeating steps 85-90 to obtain an accurate titer.
97	The total number of infected cells plated on the gradient plate is <2.5 × 106	Too little enriched zinc finger library phage was used for infection	Consider PEG precipitation to concentrate enriched zinc finger library (Box 8).
97	The total number of infected cells plated on the gradient plate is <2.5 × 106	Too little enriched zinc finger library phage was used for infection	If colonies appear to be growing on gradient plate, proceed with protocol despite the fact that the numbers were not high enough to three-fold oversample the library.
115	Failure to obtain colonies	One of the recognition helix sequences selected in the zinc finger expression plasmid contains a PstI site	Isolate zinc finger-encoding plasmid from pAC-alphaGal4 plasmid by an alternative method. Dilute undigested plasmid isolated in step 98 by 100-fold in water and transform into chemically competent XL-1 Blue cells. Patch streak transformants onto LB/TC and LB+30mg/ml chloramphenicol plates. Pick candidates that fail to grow on LB+30mg/ml chloramphenicol plates (as these were transformed only by the zinc finger expression plasmid and not the pAC-alpha-Gal4 expression plasmid) and isolate miniprep DNA. Repeat steps 114-116.
116	Failure to obtain zinc finger arrays that mediate at least 3-fold activation	Target site may not be suitable for OPEN	Consider other targets
116	Failure to obtain zinc finger arrays that mediate at least 3-fold activation	High basal level of transcription	If the absolute ß-galactosidase activity units for the Gal11P control are particularly high, consider proceeding with zinc finger arrays that activate >2-fold.