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View additional product information for GeneArt™ Seamless Cloning and Assembly Kit - FAQs (A13288)
15 product FAQs found
There is no size limit on the individual fragments as long as the combined total length of the 4 individual fragments and vector does not exceed 13 kb.
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.
Check to ensure that the cloning vector is completely linearized. Additionally, the order in 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 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.
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.
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.
If the fragments are all below 5 kb and the total size of the molecule is below 13 kb, we would recommend the GeneArt Seamless Cloning and Assembly (Plus) kit. If you are assembling elements that have no end-homology, are too large to be amplified by PCR, or are trying to create a molecule over 13 kb, we recommend the GeneArt High-Order Genetic Assembly System. Overall, the High-Order system can do more fragments, fragment editing, and oligonucleotide stitching and can do it at a higher efficiency. However, the assembly is in vivo (yeast) and takes longer to get the final construct due to longer growth periods for yeast compared to E. coli. Select the GeneArt Seamless Cloning and Assembly kit that is best for your application by visiting http://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.
This should not be a problem. An overlap that is a few bases shorter than recommended should still function in the reaction. However, for best results always use 15 bp.
The smallest insert size we have tested is 100 bp (>95% colonies contained insert). We haven't tried annealed oligos for this.
We do not recommend this as cloning efficiency/colony output can decrease. Here are some tips to increase the likelihood of a larger assembly working:
– Make sure vector background is low - RE cut the vector, gel purify, then PCR amplify the vector. If PCR not possible, you can do a second cleanup to avoid inhibition after gel purification.
– Try a ratio of 1:1 instead of 1:2.
– Do not transform more than 6 µL, and do not use OmniMAX 2 cells even though they have a higher efficiency. TOP10 and MAX Efficiency DH5alpha work best.
– Try a longer recovery time (2 hours) after addition of SOC. Use 950 µL SOC, incubate for 2 hours at 37 degrees C, and then spin down the cells. Remove ~800 µL and plate the rest on one plate.
– Longer overlaps (80 nt, for example) are better for large constructs. If the fragment ends have long overlaps, it may work better to try incubating for 45 min - 1 hour. However, small fragments (300 bp) may be negatively affected by this longer incubation - the enzyme will chew back the ends too much.
We don't recommend over-incubating since the enzyme mix may chew back too much, resulting in deletions. Shorter incubation times (e.g., 20 min) may be okay. For 4 fragments and 1 vector, we have tried 15 degrees C, RT, and 30 degrees C, and the best results were at RT with 77% cloning efficiency. The other temperatures gave us 31% and 37% efficiency. We do not recommend incubating on ice as you may get a lot of deletions at the junctions.
We suspect that there would be some degree of preference for shorter fragments. We have seen 100% cloning efficiency with a 5 kb fragment, but the colony output was lower when compared to a 2 kb fragment. For example, you get about 400 colonies per 1 µL reaction for 5 kb and about 1200-2000 colonies per 1 µL for 2 kb. Also, we have observed in assemblies of larger fragments like 5 kb that if the PCR reaction of the 5 kb fragment is not gel-purified and there is a significant PCR band at a smaller size, then the smaller fragment tends to go in more than the 5 kb. We have not observed anything like this in fragments of 1 kb or 2 kb.
In theory, the GeneArt Seamless Cloning system can be used for library construction but we have not tested either application. Adapters with the required homology to the cloning vector would have to be generated.
We recommend at least 15 bp of homology for your GeneArt Seamless cloning and we have also tried 40 or 80 bp for larger inserts.
The GeneArt Seamless PLUS kit is an improved version of the original kit. It is recommended for the assembly of up to 4 fragments and a vector totaling up to 40 kb compared to 13 kb for the original. The GeneArt enzyme mix is also provided as a 2X mix with buffer that can be stored at -20 degrees C instead of -80 degrees C. Finally, the kit comes with the linearized pYES7L vector and Stbl3/pRK2013 cells that allows for horizontal transfer of the construct into a variety of recipient strains. To select the GeneArt Seamless Cloning and Assembly kit that is best for your application, please visit https://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.