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View additional product information for GeneArt™ High-Order Genetic Assembly System - FAQs (A13285)
18 product FAQs found
The CSM Media is available as a standalone item (Cat. No. A13292).
The Lysis buffer is only available as part of the kits and is not offered as a standalone item.
There is no size limit on the individual fragments as long as the combined total length of the 10 individual fragments and vector does not exceed 110 kb.
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 µL of diluted yeast lysate in a 50 µL PCR reaction.
Ensure that yeast transformations are incubated at 30 degrees C for 3 days for proper colony formation.
Please review the following suggestions:
– Perform transformation exactly as described in protocol.
– Do not freeze-thaw or vortex MaV203 yeast competent cells.
– Use CSM-Trp agar plates for the transformation.
– For best results, use fresh DMSO from an unopened bottle. You may use DMSO stored at -20 degrees C.
Yes, you should be able to adapt your E. coli vector into a yeast-compatible cloning vector using the GeneArt High-Order Vector Conversion Cassette (Cat. No. A13291) for use with the GeneArt High-Order Genetic Assembly System with the following provisions:
– Start by using the DNA Oligo Designer web tool, and verify that your vector and the GeneArt High-Order Vector Conversion Cassette do not share internal homology to prevent potential re-arrangements when using your adapted vector with the GeneArt High-Order Genetic Assembly System.
– Use a vector with a single- or low-copy-number origin for a final construct of >15 kb, if the final plasmid construct will be transferred into E. coli. Usually, low-copy-number E. coli vectors have significantly higher capacity than high-copy number vectors.
– Avoid chloramphenicol selection markers on the custom vector since this is the marker on the cassette.
– After ligation (1:10 vector: insert ratio recommended), transform competent E. coli cells with the ligation mixture and plate on double selection LB plates (chloramphenicol plus the antibiotic marker on your custom vector backbone).To linearize your yeast-adapted cloning vector for multi-fragment assembly, a double-digestion is required to avoid background caused by residual palindromic end sequences resulting from a single enzyme digestion.
Stitching oligonucleotides used for insertion editing must have a 30-nucleotide overlap with each adjacent fragment in addition to the insertion bases (for a total length of up to 80-mer, including up to 20 insertion bases). See manual for diagram. Note: This applies for a 2-fragment assembly and the insertion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
Stitching oligonucleotides used for deletion editing must have a 40-nucleotide overlap with each adjacent fragment, annealing up to 6 nucleotides from the junction into each fragment, thus leaving up to 6 bp at the end of each fragment to be deleted during transformation-associated recombination. See manual for diagram. Note: This applies for a 2-fragment assembly and the deletion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
We recommend using our Platinum PCR Supermix High Fidelity enzyme (Cat. No. 12532-016) for amplifying DNA fragments up to 5 kb for general assembly applications. If you require higher PCR fidelity, we would recommend one that has processing and proof-reading capabilities.
Large fragments (>5 kb) are more susceptible to damage in a gel extraction procedure. Therefore, when using DNA fragments generated by PCR, we recommend that you assemble multiple fragments of less than 5 kb in one reaction rather than a single large fragment. Additionally, try to minimize UV exposure of the DNA and/or limit EtBr amount used. If you are attaching the insert to the linearized cloning vector, all nucleotides providing the requisite homology must be on the 5' end of the primer. If you are connecting two adjacent inserts, you may split the 30- to 50-bp homology between the fragments (e.g., 15 bp on the reverse primer of fragment 1 and 15 bp on the forward primer of fragment 2 for a 30-bp homology, or 25 bp on the reverse primer of fragment 1 and 25 bp on the forward primer of fragment 2 for a 50-bp homology). Please note, you can split the homology between adjacent fragments in any combination (e.g., 15 + 15 as in the example above or 20 + 10, 25 + 5 etc. for a 30-bp homology).
The pYES1L vector, a 9.4 kb linearized YAC-BAC shuttle plasmid, is a control vector for the assembly of DNA fragments in MaV203 cells and for the transfer of the assembled recombinant DNA molecule into E. coli. You can also use the pYES1L vector as a cloning vector, if desired. The origin of replication for yeast is chromosome II's centromere (single copy, high capacity YAC). The origin of replication for E. coli is F' ori (single copy, high capacity BAC). Spectinomycin is used for selection in E. coli. Four reactions (1 tube with 8µL) of the pYES1L vector are included with the kit. This is enough for control reactions, for cloning your inserts, or a combination of both. The pYES1L vector is also available separately (Cat. No. A13287, 10 reactions).
No, you can use enzymes that leave blunt ends or A-overhangs. Cloning efficiency will not be affected.
Cloning efficiencies can vary greatly based on the number of fragments with end-homology. Below are the approximate cloning efficiencies of assembled fragments into pYES1L:
>90% for 5 DNA fragments of 10 kb each
>90% for 10 DNA fragments of 5 kb each
>50% for 10 DNA fragments of 10 kb each
For pre-existing fragments without end-homology assembled into pYES1L using ‘stitching' DNA oligonucleotides, common cloning efficiencies are:
>90% for 1 fragment of 10 kb
>75% for 2 DNA fragments of 10 kb each
The web tool is not set up to do fragment editing automatically, but it can be used to do it manually. The manual has a section on fragment editing that gives examples on how to use the tool for this purpose.
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.
Yes, the kit can assemble up to 5 fragments and 3 oligo stitches.
We have developed the kit using desalted oligonucleotides, but higher purities may result in slightly better cloning efficiencies, especially during fragment editing.
We recommend having a total overlap of 50 nucleotides with adjacent fragments for constructs larger than 60 kb. For constructs smaller than 60 kb, a total overlap of 30 nucleotides is recommended.