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Invitrogen™ GeneArt™ High-Order Genetic Assembly Systems, with yeast growth media
Description
- Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
- Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
- Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
- Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
- Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments
For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt™ Seamless Cloning and Assembly Kit (cat # A13288).
Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt™ High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.
Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction step yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit. For selection of recombinant yeast clones we offer a selective yeast media kit, CSM Media for Mav203 Yeast Cells (cat# A13292).
Considerations for Choosing a Cloning Vector
The GeneArt™ High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt™ pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt™ High-Order Vector Conversion Cassette (cat#A13291).
Cloning Efficiency
In GeneArt™ High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.
Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt™ pYES1L Vector with Sapphire Technology™ are the following:
- >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
Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt™ pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
- >90% for 1 fragment of 10 Kb
- >75% for 2 DNA fragments of 10 Kb each
Design Your Cloning in silico
A key step in GeneArt™ High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI™ software.
Applications
The GeneArt™ High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Specifications
| Bacterial or Yeast Strain | MaV203, TOP10 |
| Cloning Method | Seamless Cloning |
| Content And Storage | The system has enough reagents for 10 assembly reactions and a control as well as the reagents to make 2 liters of yeast growth media. The components are: One Shot™ MaV203 yeast competent cells, One Shot™ TOP10 Electrocompetent E.coli cells, PEG⁄Lithium Acetate solution, S.O.C. medium, control cloning vector and insert for DNA assembly, supercoiled vector controls for transformation, lysis buffer, glass beads and two CSM agar media pouches and 2 bottles of sterile liquid 20% glucose to make 2 liters of CSM agar media. A cloning vector is not included in this kit. For your convenience, this product is also available without the yeast agar media under the catalog number A13285. Store the -80°C module at -80°C and the rest of the reagents at room temperature (CSM-Trp media, 20% glucose, lyses buffer, glass beads and S.O.C. medium). |
| Description | GeneArt High-Order Genetic Assembly System, with yeast growth media |
| Format | Kit |
| Sample Type | DNA |
| For Use With (Application) | Cloning |
| Number of Fragments | Up to 10 Fragments |
| Product Line | GeneArt |
| Product Type | Genetic Assembly System Kit |
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Frequently Asked Questions (FAQs)
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.
For Research Use Only. Not for use in diagnostic procedures.
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