TABLE 2.

Troubleshooting table.

Step	Problem	Cause	Solution
4A(iii)	PCR produces an extra (higher) band	Primer may be self-priming	Increasing the primer concentration to 0.6 µM (3 µl) and lowering the template to 0.01 or 0.005 ng can improve yield of the 131-bp product. The additional band does not interfere with subsequent cloning, as it is removed during gel purification
13	No colonies on the ligation plate	Linkers are not phosphorylated	Order phosphorylated linkers for cloning or phosphorylate using T4 polynucleotide kinase
		Vector is not digested with both enzymes	Check that both enzymes cleave the vector by single-digest controls
		CIP is not removed from vector DNA	Purify linearized vector backbone using Qiagen PCR purification kit
	Too many colonies on the control plate	Vector backbone is not fully digested/uncut DNA present	Repeat the XhoI/EcoRI digest, check the cutting efficiency of each enzyme individually by control single digests
		Vector backbone is not dephosphorylated	Repeat the CIP treatment, use fresh CIP enzyme
	Multiple shRNA cassettes in the miR30 vector	Concentration of 110-bp shRNA fragment too high in the ligation	Repeat the ligation and reduce shRNA fragment (insert) concentration. Do not increase insert/vector molar ratio above 5:1
50	ES cell electroporation efficiency is low	Low-quality DNA	Wash DNA thoroughly with 70% (vol/vol) ethanol (at least three times) to remove excess salts before resuspending in H2O
		Reduced cell viability	Ensure that the ES cells to be transfected are subconfluent (70–80%) and have fresh medium 4–6 h before electroporation. Keep cells on ice before and immediately after electroporation
52	No clones survive selection	Failure to induce RMCE in the ES cells	Sometimes this is due to a lack of FlpE expression. Check the quality of the pCAGs-FlpE plasmid and repeat electroporation and selection
53	ES cells differentiate	Poor-quality feeder cells	Prepare new irradiated feeder cells; make sure they are growing exponentially when irradiated and frozen. Do not use feeders that have been sitting in the incubator for more than 10 d
		Medium is exhausted	Do not let cells become overconfluent (> 90%). Ensure medium is changed daily
67	Clones do not induce GFP	Nontargeted clones	Do not pick clones that develop late in hygromycin selection. Clones that reach picking size only after 12–14 d of selection are usually not correctly targeted and do not induce GFP after dox treatment
78	ES clones do not produce viable founder animals	Bad/differentiated ES cell clone	Repeat the injection for transgenic production using a different ES clone. Ensure that the ES cell clone grows well in culture and does not show signs of marked differentiation (Fig. 5b). Check chromosomal integrity (and ploidy) of the ES clone; more than 75% should show a normal karyotype
			Use two-inhibitor medium. Although we have not required the use of 2i (MEK and GSK3 inhibitor) medium, others have shown that it can substantially improve the likelihood of obtaining tetraploid-derived founder animals29