Table 2.

Troubleshooting

Problem	Possible Cause	Solution
pENTR or pRosa26-DEST vectors do not grow	Inappropriate E. coli used Vector stock mutated	Make sure to propagate vectors in ccdB-resistant E. coli. Procure new vector stock.
No PCR product obtained when cloning into pENTR	Inappropriate annealing temperature	Perform a gradient PCR to identify the appropriate annealing temperature. Primers may be long and may require higher temperatures.
No colonies observed when cloning into pENTR	Incomplete digestion of pENTR vector PCR product sequence is incorrect	Digest with fresh restriction enzyme or for a longer period of time. Verify complete linearization using gel electrophoresis. Sequence the PCR product to ensure that In-Fusion adapters or restriction enzyme sites are successfully added.
No colonies observed after the Gateway LR reaction	pRosa26-DEST or pENTR-GOI vectors not pure LR enzyme is old LB-Amp plates not incubated for long enough pENTR-GOI is mutated	Check the quality of the DNA using a spectrophotometer and re-purify if necessary. Repeat reaction with fresh enzyme. Keep plates in the incubator until the end of the day. Sequence both the insert and portions of the pENTR backbone to ensure vector integrity.
Insufficient DNA obtained from maxi prep	Insufficient culture volume	Between 400 – 500 mL of culture may be necessary. Use extra resuspension, lysis, and neutralization solution and filtration columns if necessary.
Insufficient DNA obtained from 96-well plate during ES cell screening	DNA lost during inversion of plate	Invert the plate very slowly onto paper towels. If DNA is still lost, solution can be aspirated from the wells individually.
No bands obtained during PCR screening of ES cell clones	Inappropriate Taq polymerase or thermal cycling conditions	Use a hot start Taq polymerase and optimize thermal cycling conditions in your laboratory.
Poor signal on Southern blot	Probe specific activity is low	Use fresh [α 32P]dATP and check the specific activity of the probe via scintillation counting.
Bands not easily discernable on Southern blot	Shearing of DNA during preparation Incomplete digestion of DNA Gel not run long enough Membranes not washed sufficiently Intensifying screens not using when exposing membrane to film	Handle DNA carefully and avoid pipetting up and down to prevent random shearing of DNA. Incubate DNA with enzyme for longer to ensure complete digestion. Run gel at a low voltage for a long period of time. The bands of the DNA ladder should be well separated. Wash membranes until activity is localized on the membrane. Prewarm the wash buffers. Use intensifying screens in cassettes when exposing membranes to film, and store at −80°C during exposure.