Gateway™ pDONR™221 Vector

– 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' episome, e.g. DH10B, 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.

Typically when both large and small colonies are produced following BP recombination, we recommend screening several of both of the small and large colony types by analytical restriction digest, PCR with gene specific primers, or by sequencing to determine what you have and decide on the best course forward. If one of the colonies you analyze contains your desired entry clone, then you may proceed with this entry clone to an LR reaction to produce your desired expression clone.

There are several possible causes of this issue:

– Plasmid was lost during culture due to large size or toxicity – To improve results next time you perform a BP reaction with a tricky insert, you may try incubating your transformation plate at 30 degrees C and/or use Stbl2 E.coli to stabilize the plasmid. We also recommend performing the BP reaction positive control pEXP7-Tet alongside your BP cloning reaction of interest; the results of that control reaction will let you know whether the large and small colony phenotype is specific to your insert of interest.

– Deletions (full or partial) or point mutations in the ccdB gene – A negative BP reaction control (with no BP clonase added) should not produce any colonies – If a no BP clonase negative control produces colonies, then the ccdB/chlorophenicol cassette is compromised and we recommend to obtain a new pDONR vector.br/>
– Background on antibiotic selection plate due to contamination or expired antibiotic – If you run a no plasmid added transformation negative control plate, then this plate should not produce any colonies. Colonies on transformation negative control plate suggests contamination or need to make fresh plates.

– 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.
– Check primer design and try gel/PEG purifying the attB-PCR product.
– If the attB-PCR product or linear attB Expression clone is too long (>5 kb), incubate the BP reaction overnight.

You may continue, but the recombination efficiency will drop by approximately 10 fold.