Gateway™ LR Clonase™ II Enzyme mix

– Check whether the reaction was transformed into an E.coli strain containing the F' episome and the ccdA gene – use an E.coli strain that does not contain the F&339; episome, e.g. OmniMAX 2-T1R, TOP10.
– Deletion (full or partial) of the ccdB gene – propagate in media with 50-100 mg/mL ampicillin and 15-30 µg/mL chloramphenicol.
– Contamination from another resistant strain.
– Check whether proper amount of DNA was used in the reaction.

– Plasmid was lost during culture due to large size or toxicity – try incubating at 30 degrees C; use Stbl2 E.coli to stabilize the plasmid.
– Deletions (full or partial) or point mutations in the ccdB gene – obtain a new Destination vector.
– Small colonies may be unreacted entry clone that co-transforms with the Expression clone – reduce the amount of Entry clone to 50 ng per 10 µL reaction; reduce the volume of sample used for transformation to 1 µL; for a Destination vector with ampicillin selection marker, increase the ampicillin concentration to 300 µg/mL.

– Check the competent cells with pUC19 transformation.
– Increase the amount plated.

– Increase the incubation time up to 18 hours.
– Make sure to treat reactions with proteinase K before transformation.
– Check whether the correct antibiotic was used for selection.
– Check whether the att site sequences are correct.
– Check whether the correct Clonase enzyme was used and whether it was functional.
– Check whether the recommended amount of DNA was used in the reaction.
– Perform the positive control recombination with pENTR-Gus plasmid.
– If the Entry clone or Destination vector is too large (>10 kb), incubate the LR reaction overnight, linearize the Destination vector or the Entry clone or relax the Destination vector with topoisomerase I.

No, since a stop codon would be necessary for an N-terminal tagged destination vector, whereas the presence of a stop codon would block expression of the C-terminal tag.