Invitrogen™ GeneArt™ pYES1L Vector with Sapphire™ Technology

Catalog No.	Quantity
A13287	10 Reactions

Catalog No. A13287 Supplier Invitrogen™ Supplier No. A13287

Description

A ready-to-use, 9.3 Kb linearized BAC/YAC shuttle vector, used for the assembly of DNA fragments with the GeneArt™ High-Order Genetic Assembly System

GeneArt™ pYES1L Vector with Sapphire Technology™ is a ready-to-use, 9.3 Kb linearized BAC⁄YAC shuttle vector, used for the assembly of DNA fragments with the GeneArt™ High-Order Genetic Assembly System (cat#A13285 & A13286). The vector has the capacity to clone up to 110 Kb of DNA fragments. This product only includes the linearized GeneArt™ pYES1L Vector with Sapphire Technology™.

Some of the key features and elements of the GeneArt™ pYES1L Vector with Sapphire Technology™ include:

• TRP1 to allow selection of yeast transformants in tryptophan-deficient medium
• ARS⁄CEN origin to allow stable maintenance of the construct in yeast
• F’ for conjugation
• oriT for maintenance of large constructs in E.coli
• Spectinomycin resistance for selection in E.coli
• 6 Convenient and validated restriction sites flanking the cloning region

For Research Use Only. Not for use in diagnostic procedures.

Specifications

• Antibiotic Resistance Bacterial: Spectinomycin
• Cloning Method: Seamless Cloning
• Content And Storage: The product contains 3 vials of linear GeneArt pYES1L Vector with Sapphire Technology at a concentration of 50 ng/mL. Vials contain enough plasmid for 10 cloning reactions using the GeneArt High-Order Genetic Assembly System (needs to be purchased separately).
  
  Store the product at -80°C.
  
  The vector is guaranteed stable for 6 months when properly stored.
• Sample Type: DNA
• For Use With (Application): Cloning
• Number of Fragments: Up to 10 Fragments
• Product Line: GeneArt
• Quantity: 10 Reactions
• Replication Origin: ARS⁄CEN, oriT
• Selection Marker Eukaryotic: TRP1
• Shipping Condition: Dry Ice
• Size: 110 kb total (vector plus all inserts)
• Vector: pYES1L Linearized

Frequently Asked Questions (FAQs)

With the GeneArt High-Order Genetic Assembly System, I'm getting no positive colonies detected by yeast colony PCR. Can you please offer some troubleshooting tips?

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.

With the GeneArt High-Order Genetic Assembly System, I see small or no yeast colonies after transformation. Can you please offer some suggestions?

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

With the GeneArt High-Order Genetic Assembly System, I did not get any yeast colonies after transformation and the transformation control did not work. Can you please offer some suggestions?

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.

I want to use my own E. coli vector for my assembly. Can I do this? How?

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.

With the GeneArt High-Order Genetic Assembly System, what are the requirements for stitching oligonucleotides used for insertion editing versus deletion editing?

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 the GeneArt High-Order Genetic Assembly System, what enzyme do you recommend for amplifying my DNA fragments?

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.

I am using the GeneArt High-Order Genetic Assembly System and am trying to assemble large pieces of DNA. Do you have any recommendations?

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).

What are the specifications for the pYES1L vector? Can I use this as my cloning vector?

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).

Will the addition of A-overhangs by the PCR enzyme affect cloning efficiency with the GeneArt High-Order Genetic Assembly System?

No, you can use enzymes that leave blunt ends or A-overhangs. Cloning efficiency will not be affected.

What cloning efficiency can be expected using the GeneArt High-Order Genetic Assembly System?

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

How do I use the web-based tool for fragment editing with the GeneArt High-Order Genetic Assembly System?

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.

I'm cloning 4 DNA fragments in a vector. Should I use the GeneArt Seamless Cloning and Assembly (Plus) Kit or the GeneArt High-Order Genetic Assembly Kit?

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.

With the GeneArt High-Order Genetic Assembly System, can I assemble a molecule with fragments that carry homology (PCR) and others that are pre-existing (stitching) at the same time?

Yes, the kit can assemble up to 5 fragments and 3 oligo stitches.

Do you recommend a particular purity of oligonucleotides for the GeneArt High-Order Genetic Assembly System?

We have developed the kit using desalted oligonucleotides, but higher purities may result in slightly better cloning efficiencies, especially during fragment editing.

With the GeneArt High-Order Genetic Assembly System, how many overlapping nucleotides must I have if my construct is larger than 60 kb?

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.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.