TABLE 1.

Troubleshooting table.

Step	Problem	Solution
20	No RNA synthesis	Sequence the ZFN expression vector to make sure that it contains the insert
	Poor RNA synthesis or RNA smear on the gel	Make sure the RNA transcription kit is not expired and use RNase-free reagents
28	Too many dead or deformed embryos after RNA injection	For most ZFN pairs, we observe 30–80% of dead/deformed embryos when injected with 250 ng/μl of ZFN-encoding RNAs; if the toxicity of a ZFN pair is too high, reduce the RNA concentration to 100–150 ng/μl
	No dead or deformed embryos after RNA injection	Make sure that the solution is injected into the cytoplasm at the 1-cell stage; if that’s the case, increase the RNA concentration 2 to 3 fold
37	Many clones do not contain PCR inserts	Make sure that the PCR worked, and/or that the TOPO cloning kit is not expired
	No somatic mutation is found by sequencing	If no somatic mutation is found after obtaining 96 sequences of the PCR inserts, try to inject a higher concentration of ZFN-encoding RNAs or use a different pair of ZFNs
38	Too few zebrafish survive to adulthood	Repeat the injections at a 2-fold lower RNA concentration and raise the injected embryos
52	Too many fluorescent PCR fragments amplified from the control sample	Adjust the PCR conditions or design new pairs of gene-specific primers
61	Incomplete restriction enzyme digestion of the control sample	Some enzymes may be less efficient than the others; increase the volume of the digestion reaction and use more of the enzyme
	No founders are identified using restriction digestion analysis	Some insertional mutations may not affect the restriction enzyme recognition sites; screen more F0 fish or try to identify founders using the other two methos