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Invitrogen™ GeneArt™ Seamless Cloning and Assembly Kit

Catalog No. A13288
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Catalog No. A13288 Supplier Invitrogen™ Supplier No. A13288
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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

The GeneArt™ Seamless Cloning and Assembly Kit 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. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.
  • Speed and Ease — Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); 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
  • Vector Flexibility — Use our linear vector or a vector of your choice
  • Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
  • Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning more than 4 DNA fragments, final molecules larger than 110 Kb, or for using pre-existing DNA elements too long to be amplified by PCR; please consider the GeneArt™ High-Order Genetic Assembly System (cat# A13285).

Simple and Fast Clone Creation
GeneArt™ Seamless Cloning is a simple, two step process, consisting of a tube based assembly reaction followed by transformation into One Shot™ Chemically Competent TOP10 E. coli. The kit uses the properties of a proprietary enzymatic mix to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). A portion of the assembly reaction is then transformed into the provided competent TOP10 cells, yielding clones ready for analysis the next day. The required 15-base pair end-homology can be easily engineered by PCR-amplification with custom DNA oligos.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt™ Seamless Cloning and Assembly Kit the main factors effecting cloning efficiency are the size of the DNA elements (100 bp to 5 Kb), the total size of the final molecule (≤ 13 Kb), 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 cloned into pUC19L are the following:
  • 90% for a single 5 Kb DNA element
  • 70% for 4 fragments of 1 Kb each
  • 40% for 4 DNA fragments of 2 Kb each

Success of the cloning 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 by PCR. The circularized clones obtain 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 to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos with end-homology for the PCR amplification 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™ Seamless Cloning and Assembly Kit 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 in an existing vector, and many other techniques that require manipulation of genetic sequences.

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

Specifications

Bacterial or Yeast Strain TOP10
Cell Type Chemically Competent E. coli
Cloning Method Seamless Cloning
Content And Storage The kit has enough reagents for 20 cloning reactions and a control. The components are: 10X Enzyme Mix and 5X Reaction Buffer, control vector and insert, One Shot™ Chemically Competent TOP10 E. coli, S.O.C. Medium, and a supercoiled control plasmid for E.coli.

A cloning vector is not included in this kit.

For your convenience and flexibility, a ready-to-use linear vector is available separately for 20 cloning reactions (GeneArt™ Linear pUC19L Vector for Seamless Cloning, catalog number A13289).
Format Kit
For Use With (Application) Cloning
Number of Fragments Up to 4 Fragments
Product Line GeneArt
Product Type Cloning and Assembly Kit
Quantity 20 Reactions
Size 13 kb total (vector plus all inserts)
Vector pUC19L Linearized
Workflow Step DNA Assembly
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Frequently Asked Questions (FAQs)

In the GeneArt Seamless Cloning and Assembly kit, is there a size limit for the 4 individual fragments that I can clone into the vector?

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.
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 used electrocompetent cells instead of chemically competent cells with the GeneArt Seamless Cloning and Assembly Kit and now my reaction isn’t working. Why is this?

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.
I accidentally stored my GeneArt Seamless Cloning and Assembly enzyme mix at -20 degrees C instead of -80 degrees C. Is it still okay to use?

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.
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.