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Invitrogen™ GeneArt™ Seamless PLUS Cloning and Assembly Kit

Catalog No. a14603
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Description

The complete kit for simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute or less, room temperature reaction

Like our first generation Seamless Cloning and Assembly Kit, GeneArt™ Seamless PLUS Cloning and Assembly Kit is the complete kit for simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute or less, room temperature reaction. However, Seamless PLUS offers several advantages over previous kits:

• Increased Efficiency: pre-cloning option for large fragments for increased cloning efficiency
• Larger Constructs: create constructs up to 40 kb
• Versatility: high capacity, broad-range conjugative vector that replicates in most Gram negative bacteria

The improvements above are combined with these key benefits shared by all GeneArt™ Seamless Cloning and Assembly kits:

• Speed and Ease—clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector; 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
• Free Tools—design 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
• Diverse Applications—streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning of more than 4 DNA fragments or for final molecules larger than 110 Kb, please consider the GeneArt™ High-Order Genetic Assembly System.

Simple and Fast Clone Creation
GeneArt™ Seamless PLUS Cloning is a simple, two-step process, consisting of in vitro assembly followed by transformation into One Shot™ DH10B™ T1R SA competent E. coli. The kit employs a proprietary enzyme/buffer mix to assemble DNA fragments with shared terminal end homology without extra sequences or scars in the final construct (“seamless”). Terminal end homology is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt™ Seamless PLUS Cloning and Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA fragments (100 bp to 10 Kb), the total size of the final molecule (≤ 40 Kb), and the quality and specificity of each fragment.

Typical cloning efficiencies for different numbers of fragments:

• >95% for 4 fragments, 5 Kb each
• >90% for 4 fragments, 10 Kb each

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 Design Support
A key step in GeneArt™ Seamless PLUS Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. 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 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 PLUS Cloning and Assembly Kit 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 kit 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.

Specifications

Bacterial or Yeast Strain DH10B
Cell Type Chemically Competent E. coli
Cloning Method Seamless Cloning
Content And Storage GeneArt Enzyme Mix (2X)
pUC19L Linearized Vector
pYES7L Linearized Vector
2 control inserts
One Shot™ DH10B™ T1R SA Cells
pUC19 Control
Stbl3â„¢/pRK2013 Glycerol Stock
S.O.C. medium
Format Kit
For Use With (Application) Cloning
Number of Fragments Up to 4 Fragments
Product Type Cloning and Assembly Kit
Quantity 20 Reactions
Size 40 kb total (vector plus all inserts)
Vector pUC19L Linearized, pYES7L Linearized
Workflow Step DNA Assembly
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Frequently Asked Questions (FAQs)

In the GeneArt Seamless PLUS 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 40 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'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 unique about the One Shot DH10B T1 SA cells that come in the GeneArt Seamless PLUS Cloning and Assembly Kit? Can other cells be substituted?

The DH10B T1 SA cells are optimized for cloning large assemblies and we don't recommend substitution with other competent cells.
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.