I performed a BP reaction and got high background after transformation. Can you please offer some troubleshooting tips?

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Answer

– 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. 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.

Answer Id: E6851

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Can I use pDONR201 and pDONR207 as Donor vectors?

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Answer

pDONR201 and pDONR207 can be used, but they replicate less efficiently than the Donor vectors we offer, pDONR221 and pDONR/Zeo, even though both pDONR201 and pDONR207 contain the pUC ori. This results in lower plasmid yields. With pDONR221, plasmid yields are in the range of 0.5 - 1.0 µg of DNA per mL of culture.

Answer Id: E6800

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How much DNA should I use in my BP reaction?

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Answer

For the most efficient BP reaction, it is best to not have attB sites in molar excess of attP sites. The standard BP Reaction (20 µL) uses 300 ng (no more than 500 ng) of pDONR vector and 30-300 ng attB-flanked PCR product or Expression Clone for 1 hour at 25 degrees C. Using too much of the Donor vector in the reaction tube will inhibit the BP reaction and also result in intact donor vector being co-transformed with the Entry Clones. This will reduce the number of colonies on the plate by killing the transformed E. coli due to the presence of the ccdB gene. Longer incubation times of up to 24 hours can be used to convert a higher percentage of starting attB-DNA to product. For PCR products > 4 kb, the number of colonies obtained per fmol of PCR DNA added decreases with increasing size. Thus, for larger PCR products, it is recommended to increase the amount of DNA to at least 100 fmol of PCR product per 20 µL reaction, and using incubations longer than one hour (e.g., 6 hours or overnight to 24 hours). The largest PCR-amplified DNA cloned in-house was 10.1 kb. Increasing the incubation to 4-6 hours will typically increase colony output 2-3 fold and 16-24 hours will typically increase colony output 5-10 fold.

Answer Id: E6802

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I performed a BP reaction and got few or no colonies after transformation, whereas the transformation control gave colonies. Can you please offer some suggestions?

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Answer

– 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.

Answer Id: E6845

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What is the difference between your pDONR221 and pDONR/Zeo vectors?

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Answer

The backbones of the pDONR221 and pDONR/Zeo vectors are exactly the same except for the antibiotic resistance marker. pDONR221 has the kanamycin antibiotic resistance marker, while pDONR/Zeo has the zeocin resistance marker.

Answer Id: E6801

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I performed a BP reaction and got two distinct types of colonies (large and small) after transformation. How should I proceed?

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Answer

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.

Answer Id: E6849

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I performed a BP reaction and got no colonies after transformation, and the recombination positive control was not successful. Can you please offer some suggestions?

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Answer

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

Answer Id: E6847

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I forgot to add proteinase K to my BP reaction. Can I continue?

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Answer

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

Answer Id: E6803

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