Invitrogen™ GeneArt™ Seamless Cloning and Assembly Enzyme Mix

Catalog No.	Quantity
A14606	20 Reactions

Catalog No. A14606 Supplier Invitrogen™ Supplier No. A14606

Description

Enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction

GeneArt™ Seamless Cloning and Assembly Enzyme Mix enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. GeneArt™ Seamless enzyme mix is the economical choice for creating constructs up to 13 kb with the option for high-throughput assembly.

• Efficiency: pre-cloning option for large fragments for increased cloning efficiency
• Speed and Ease: clone DNA fragments, with sequence of your choice, in a single vector (up to 13 kb); no restriction digestion, ligation, or recombination sites required
• Free Tools: design of your final construct and DNA oligos in silico using our free web-based tool that takes you step-by-step through your project
• Vector Flexibility: use our linear vector or a vector of your choice

For the cloning of pre-existing DNA elements too long to be amplified by PCR and for final molecules up to 40 Kb, please consider the GeneArt™ Seamless PLUS Cloning and Assembly Kit (1–4 DNA fragments only); and for final molecules larger than 110 Kb, please consider the GeneArt™ High-Order Genetic Assembly System.

Simple and Fast Clone Creation
GeneArt™ Seamless Cloning is a simple, two step process, consisting of in vitro assembly followed by transformation. The enzyme mix uses a proprietary enzyme/buffer mix to assemble DNA fragments without extra sequences or scars (“seamless”). Terminal end homology, if needed, is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

Cloning Efficiency, Flexibility, and Precision
Cloning success is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or PCR. The circularized clones obtained from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Cloning Design Support
A key step in GeneArt™ Seamless Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. To simplify and speed the design process, we provide a free online tool, the GeneArt™ Design Tool for Seamless or High-Order Assembly and Mutagenesis, to help you design your experiment in silico. The tool guides the user through construct design, including fragment import, order, and orientation; checks for areas of homology and potential design issues; creates primers for pre-cloning or incorporating end homology between fragments, if needed; and generates final construct map files and sequence in Genbank format for download or import into Vector NTI™ software.

Applications
The GeneArt™ Seamless Cloning and Assembly Enzyme Mix is designed to empower cloning and DNA assembly 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 mix to construct fusion proteins; delete, replace, or add DNA elements such as restriction sites in an existing vector; and carry out many other techniques that require manipulation of genetic sequences.

For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.

Specifications

• Cloning Method: Seamless Cloning
• Content And Storage: 1 vial GeneArt Enzyme Mix (2X)
  pUC19L Linearized Vector
  Control Insert
• Format: Kit
• For Use With (Application): Cloning
• Number of Fragments: Up to 4 Fragments
• Quantity: 20 Reactions
• Size: 13 kb total (vector plus all inserts)
• Vector: pUC19L Linearized
• Workflow Step: DNA Assembly

Frequently Asked Questions (FAQs)

With the GeneArt Seamless Cloning and Assembly Kit, a large number of the transformants contain the incorrect insert. Can you please offer some suggestions?

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.

With the GeneArt Seamless Cloning and Assembly Kit, a large number of the transformants contain no insert. Can you please offer some suggestions?

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.

With the GeneArt Seamless Cloning and Assembly Kit, I'm getting no colonies after transformation with DNA inserts, but the transformation with control assembly reaction is successful. Can you please offer some troubleshooting tips?

– 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.

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.

I am using the GeneArt Seamless Cloning and Assembly Kit, and I accidentally designed my primers so there is only a 14 bp overlap. Is this acceptable?

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.

With the GeneArt Seamless Cloning and Assembly Kit, what is the smallest insert size you have tested, and is it possible to use annealed oligos for the insert?

The smallest insert size we have tested is 100 bp (>95% colonies contained insert). We haven't tried annealed oligos for this.

Is it possible to perform assemblies larger than 13 kb total with the GeneArt Seamless Cloning and Assembly kits?

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.

With the GeneArt Seamless Cloning and Assembly kits, what happens if the cloning reaction is incubated for shorter or longer periods than the recommended 30 min at RT? What happens at lower temperatures?

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.

Is the cloning efficiency with the GeneArt Seamless Cloning and Assembly kits different between shorter and longer fragments?

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.

Can the GeneArt Seamless Cloning system be used for library construction? What about for cloning of random DNA libraries made from oligos?

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.

Could homology longer than 15 bp interfere with recombination efficiency while performing GeneArt Seamless cloning?

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.

What is the difference between the GeneArt Seamless PLUS Cloning and Assembly Kit and the original GeneArt Seamless Cloning and Assembly Kit?

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.

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