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Having difficulties with your experiment?
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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).
If the insert is potentially toxic to the host cells, here are some suggestions that you can try:
Here are possible causes and suggestions:
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).
Unfortunately, the 10X Enzyme Mix in the GeneArt® Seamless Cloning and Assembly Kit is unstable at -20 degrees C, and will quickly lose activity at this temperature. We have seen a large drop in colony output after storing the enzyme mix at -20 degrees C for 2 months.
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 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:
Please review the following suggestions:
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:
Here are some possible causes and solutions:
This indicates that the GeneArt® Type IIs Enzyme Mix was handled incorrectly. Please review the following suggestions:
We recommend using freshly prepared LB plates containing the selection antibiotic appropriate for your cloning vector.
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