Gateway Recombination and Seamless Cloning Support – Troubleshooting

• 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.

• 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.

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

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

• Plasmid was lost during culture due to large size or toxicity (you can 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 pDONR vector).

• Plasmid was lost during culture due to large size or toxicity (you can 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 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).

• Check for deletion (full or partial) of the ccdB gene (propagate in media with 50-100 mg/mL ampicillin and 15-30 µg/mL chloramphenicol.
• Check for contamination from another resistant strain.
• Check whether proper amount of DNA was used in the reaction.

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

• After the transformation, incubate at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
• Try using Stbl2 cells for the transformation.

Here are possible causes and suggestions:

• Incorrect PCR primer design: Make sure that the forward PCR primer contains the sequence, CACC, at the 5′ end. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in the Directional TOPO® vector.
• Reverse PCR primer is complementary to the GTGG overhang at the 5′ end: Make sure that the reverse PCR primer does not contain the sequence, CACC, at the 5′ end.
• Insert cloned in the correct orientation is potentially toxic resulting in cloning in the reverse orientation: After transformation, incubate the cells at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
• Try using Stbl2 cells for the transformation.

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).

• Check for deletion (full or partial) of the ccdB gene (propagate in media with 50-100 mg/mL ampicillin and 15-30 µg/mL chloramphenicol.
• Check for contamination from another resistant strain.
• Check whether proper amount of DNA was used in the reaction.

• Check whether the correct Clonase® enzyme was used and whether it was functional (make sure that LR Clonase® II Plus was used).
• Check whether the correct combination of Entry vectors and Destination vector was used.
• Ensure that the correct entry clones were used (and that they were sequenced).
• Check if the recommended amount of Entry vector and Destination vector was used in the reaction.
• Perform the LR reaction positive controls to determine which entry clone may be faulty (note that MultiSite GW 3-Fragment kit only comes with a single control vector, pMS/GW, that is used to make all control pENTR vectors).
• Check whether the correct antibiotic was used for selection.
• Check whether the att site sequences in the Destination vector are correct.
• Increase the incubation time up to 18 hours.

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

• 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).
• This may be due to deletion (full or partial) of the ccdB gene (propagate in media with 50-100 mg/mL ampicillin and 15-30 µg/mL chloramphenicol.
• This may be due to contamination from another resistant strain.
• Check whether proper amount of DNA was used in the reaction.

We do not recommend using electrocompetent cells. The enzyme mix does not perform ligation of the DNA ends, so electroporation will disrupt the DNA base pairs formed during assembly.

Please check the following:

• Check the purity of the PCR products.
• Ensure that the required end-terminal homology between ends is present.
• DNA ends may have been damaged during preparation (exposure to UV); limit exposure time to UV/EtBr.
• Check ratio and amounts of DNA inserts and vector (2:1 insert:vector molar ratio).
• Ensure that the GeneArt® Enzyme Mix is handled correctly. This enzyme is temperature sensitive. Return immediately to storage after use. Do not leave at room temperature or on ice for extended periods.

Check to ensure that the cloning vector is completely linearized. Additionally, the order of which the GeneArt® Enzyme mix is added is crucial to the experiment – add it last. Lastly, check the incubation time of the reaction for the recommended time.

Check your PCR product on a gel. You may be getting multiple products, causing incorrect inserts to clone in to your vector. Gel purification of the PCR products can help with this issue.

Please review the following suggestions:

• Make sure DNA fragments have the required end homology and the appropriate amount is added.
• If using stitching oligonucleotides, you must include both the sense and anti-sense strand (you don’t have to anneal them).
• Purify fragments with a PCR cleanup kit. If using gel-purified DNA, use extra caution to avoid damaging the DNA by minimizing exposure and using low UV power.
• Ensure that your vector has the appropriate genetic elements to propagate in yeast. If not, we would recommend using the GeneArt® High-Order Vector Conversion Cassette.

Ensure that yeast transformations are incubated at 30 degrees C for 3 days for proper colony formation.

We would recommend trying to re-streak the colony on a fresh plate and repeat colony PCR. Do not break open the yeast cells with the beads supplied with the kit; the beads are for transformation into E. coli. Additionally, use less than 0.5 microliters of diluted yeast lysate in a 50 uL PCR reaction.

Here are some possible causes and solutions:

• PCR products not pure enough: Repeat PCR amplification and purify product using a different method of purification. Alternatively, perform phenol:chloroform extraction on your original PCR product, followed by ethanol precipitation.
• DNA inserts do not contain the required recognition sequences for the Type IIs endonuclease: Make sure that your DNA inserts and the cloning vector contain the required sequences for GeneArt®Type IIs assembly reaction (refer to page 11 of the manual for sequence requirements for 5′ DNA overhangs, and page 14 for requirements on PCR primer design). Alternatively, use the GeneArt® Primer and Construct Design Tool for primer design.
• Ends of the DNA inserts generated by PCR were damaged: Employ extra caution to minimize any potential damage to the ends of your DNA inserts by leaving the gel on the gel tray when exposing to UV light, using low UV power, and minimizing the time the gel is exposed to UV light. Note that an additional isopropanol precipitation after gel purification might be required to obtain the best results.
• Incorrect amounts of DNA inserts and/or vector were used: Make sure that you use the correct amounts of DNA inserts, and/or vector for cloning. For best results, use the GeneArt® Primer and Construct Design Tool to determine the amount of each DNA fragment to use.
• Primers used for generating the DNA fragments were of low quality: Make sure to use high quality primers devoid of mutations as only a single mutation in the restriction recognition site or in the overhang sequence can be sufficient to obliterate a multi-fragment assembly.
• DNA inserts contain multiple repetitive sequences: Multiple repetitive sequences might result in lethal phenotypes.

This indicates that the GeneArt® Type IIs Enzyme Mix was handled incorrectly. Please review the following suggestions:

• Quickly thaw the GeneArt® Type IIs Enzyme Mix on ice, and immediately return to –80 degrees C after use.
• Do not subject the enzyme mix to more than 6 freeze/thaw cycles.
• Do not leave the enzyme mix at room temperature or on ice for extended periods of time.
• Use the reaction cocktail and the enzyme mix promptly; do not keep them for any extended period of time before starting the cloning reaction.

We recommend using freshly prepared LB plates containing the selection antibiotic appropriate for your cloning vector.