GeneArt Strings DNA Fragments and Libraries

GeneArt™ Strings DNA Libraries are pools of GeneArt™ Strings DNA Fragments from 200 – 2000 bp, containing up to 3 blocks of degenerate nucleotides with randomized distribution (see schematic below). Each block can consist of up to 30 bp using the full IUPAC code of DNA nucleotides (U is not optional). Blocks need to be separated by 30 bp of non-degenerate nucleotides. At least 500 ng are produced within 10-15 business days.

All GeneArt™ Strings DNA products can be designed, optimized for protein expression, and ordered within our online order portal for delivery in tube format.

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Complete usage instructions can be found in the GeneArt™ Strings Product Bulletin. Choose the PDF based on your location:

• GeneArt™ Strings product bulletin and usage instructions- USA 
• GeneArt™ Strings product bulletin and usage instructions - Canada 
• GeneArt™ Strings product bulletin and usage instructions - Rest of world outside North America 

Strings DNA products are suspended in 10 mM Tris pH 8.5 buffer and delivered dried, ready for resuspension and cloning. We recommend that you centrifuge tubes prior to opening, add the appropriate amount of water to the bottom of the tube, and incubate at least 1 hour at room temperature (alternatively incubate at 4°C overnight). After incubation, carefully resuspend Strings DNA products and use immediately. If not used immediately, resuspended Strings DNA products should be aliquoted and frozen at –20°C. Please avoid freeze-thaw cycles.

Delivery amounts:
GeneArt Strings DNA Fragments: ≥ 200 ng
GeneArt Strings DNA Libraries: ≥ 500 ng

Complete usage instructions can be found in the GeneArt Strings Product Bulletin. Choose the PDF based on your location:

GeneArt Strings product bulletin and usage instructions- USA

GeneArt Strings product bulletin and usage instructions - Canada

GeneArt Strings product bulletin and usage instructions - Rest of world outside North America

Figure 1 shows an image of nine Strings DNA Fragments and five Strings DNA Libraries resuspended and separated using agarose gel electrophoresis .

Strings DNA Fragments (lanes 1-4 and 6-10) and Strings DNA Libraries (lanes 12-16) after gel electrophoresis (1% agarose gel). Library 1 is 501 bp; Library 2 is 649 bp; Library 3 is 998 bp; Library 4 is 404 bp; and Library 5 is 1989 bp. DNA marker is 1 kb DNA Ladder.

The GeneArt production team performs a biosecurity screen on each sequence entered. In the event your order does not pass this analysis, you will be contacted to determine the best way to proceed.

Strings DNA products can be cloned using any available cloning method, just make sure to design the ends of your sequence according to the respective requirements. You can simply paste the sequence into the online order form of the web portal and proceed to the cart to order. Alternatively, you can use the convenient GeneArt portal sequence editing and optimization functions. Depending on your desired cloning system, you may need to modify the 5’ and 3’ ends of your sequence to enable proper insertion into your desired vector. For example, for cloning Strings DNA products by restriction enzymes/ligation, we generally recommend adding additional stuffer nucleotides to both ends of your sequence to ensure efficient enzyme binding. If blunt cloning methods are used (e.g., TOPO™ technology), we recommend that you add 5–10 nucleotides of random stuffer DNA on both ends of the fragment, preserving functional DNA elements needed for downstream applications (by their very nature, linear DNA fragments are not entirely free of small terminal truncations).

Table 1 lists some points to consider for frequently used cloning methods.

Table 1. Examples for cloning methods and recommended sequence design.

Strings DNA Fragments are PCR amplicons of assembled oligonucleotides, ready for screening to identify the correct clone. Strings DNA Fragments are bulk sequence-controlled as part of the QC process to help ensure that your desired sequence is present in the fragment pool.

To identify a correct clone >90% of the time, we recommend to use the screening guidelines in Table 2.

Table 2. Recommended screening of Strings DNA Fragments.

An example for a screening analysis of cloned Strings DNA Fragments is given in Figure 2.

Figure 2. Cloned Strings Fragments. Two Strings DNA Fragments (each 2640 bp) were cloned into GeneArt standard cloning vector pMA, and six colonies were analyzed by colony PCR followed by agarose gel electrophoresis. (Amplicons are longer since primer design for colony PCR adds ~ 100 bp 5’ & 3’ each)

Restriction enzyme cloning example with GeneArt Strings DNA Libraries
All 5 libraries were cloned by restriction digest, ligation and transformation of E. coli by electroporation. Resulting colony forming units (cfu) were counted and a value for the cloning of 500 ng library was extrapolated (blue bars). Colony-PCR was performed on 16 colonies each and the cloning efficiency calculated as the percentage of colonies with an insert of expected size (red bars). Error bars indicate standard error of the mean from multiple experiments. 

GeneArt Seamless Cloning and Assembly example with Strings DNA Libraries
All 5 libraries were cloned using the GeneArt Seamless Cloning and Assembly Kit with subsequent heat shock transformation of E. coli. Resulting colony forming units (cfu) were counted and a value for the cloning of 500 ng library was extrapolated (blue bars). Colony-PCR was performed on 16 colonies each and the cloning efficiency calculated as the percentage of colonies with an insert of expected size (red bars). Error bars indicate standard error of the mean from multiple experiments.

GeneArt Type IIs Assembly example with Strings DNA Libraries
All 5 libraries were cloned using the GeneArt Type IIs Assembly Kit AarI with subsequent transformation of E. coli by electroporation. Resulting colony forming units (cfu) were counted and a value for the cloning of 500 ng library was extrapolated (blue bars). Colony-PCR was performed on 16 colonies each and the cloning efficiency calculated as the percentage of colonies with an insert of expected size (red bars). Error bars indicate standard error of the mean from multiple experiments.

Restriction enzyme cloning example with GeneArt Strings DNA Fragments

Eight Strings DNA Fragments up to 3,000 bp were cloned using 5’ AscI and 3’ PacI restriction enzymes. After ligation and transformation, bacteria were plated and incubated overnight at 37°C. Eight  (<1 kb) or 16 (>1 kb) colonies were picked, and colony PCR was performed to identify full-length clones; four to eight full-length clones were sequenced to determine the number of sequence-correct clones.

The figure shows the percentage of clones with correct length as identified by colony PCR (cloning efficiency) and the percent of sequence-correct clones (fidelity) based on the number of full-length clones.

GeneArt Type IIs Assembly example with multiple Strings DNA Fragments

Eight Strings DNA Fragments of different lengths up to 2,800 bp with suitable sequence ends (AarI recognition sequence and 6 bp stuffer nucleotides) were used for direct assembly with the GeneArt Type IIs Assembly Kit Aar I (Cat. No. A15916). The resulting four genes were 5,056 bp, 5,061 bp, 5,267 bp, and 5,448 bp long. After ligation and transformation, bacteria were plated and incubated overnight at 37°C. Sixteen colonies were picked and colony PCR was performed to identify full-length clones. For all four assembled genes, eight colonies were sequenced to determine the number of sequence-correct clones.

The figure shows the percentage of clones with correct length as identified by colony PCR (cloning efficiency) and the percent of sequence-correct clones (fidelity) based on the number of full-length clones.

GeneArt Seamless Cloning example with GeneArt Strings DNA Fragments

Ten Strings DNA Fragments up to 3,000 bp were cloned using the GeneArt Seamless Cloning and Assembly Kit (Cat. No. A13288). After ligation and transformation, bacteria were plated and incubated overnight at 37°C. Eight (<1 kb) or 16 (>1 kb) colonies were picked, and colony PCR was performed to identify full-length clones; four to eight full-length clones were sequenced to determine the number of sequence-correct clones.

The figure shows the percentage of clones with correct length as identified by colony PCR (cloning efficiency) and the percent of sequence-correct clones (fidelity) based on the number of full-length clones.

GeneArt Seamless Assembly example with multiple Strings Fragments

Strings DNA Fragments are offered in lengths up to 3,000 bp. However, GeneArt Seamless assembly technology can be used to directly assemble two Strings DNA Fragments up to 1 kb without pre-cloning of the subfragments. If you want to assemble more Strings DNA Fragments or longer Strings DNA Fragments using seamless assembly technology, we generally recommend pre-cloning of the subfragments to limit the screening effort needed to find the correct clone after assembly. Alternatively, you can use GeneArt Type IIs assembly technology.

Twelve Strings DNA fragments of different lengths up to 1 kb with suitable sequence ends (15 bp homology to the vector or to the next fragment) were used for direct assembly with the GeneArt Seamless Cloning and Assembly Kit (Cat. No. A13288). The resulting six genes were 1,375 bp, 1,466 bp, 1,536 bp, 1,590 bp, 1,647 bp, and 1,853 bp long. After ligation and transformation, bacteria were plated and incubated overnight at 37°C. Sixteen colonies were picked, and colony PCR was performed to identify full-length clones. For all six assembled genes, six olonies were sequenced to determine the number of sequence-correct clones.

The figure shows the percentage of clones with correct length as identified by colony PCR (cloning efficiency) and the percent of sequence-correct clones (fidelity) based on the number of full-length clones.

Gateway cloning example with GeneArt Strings DNA Fragments

Five Strings DNA Fragments up to 1,000 bp with attB sites were cloned into pDONR™221. After ligation and transformation, bacteria were plated and incubated overnight at 37°C. Eight colonies were picked, and colony PCR was performed to identify full-length clones; four to seven full-length clones were sequenced to determine the number of sequence-correct clones.

The figure shows the percentage of clones with correct length as identified by colony PCR (cloning efficiency) and the percent of sequence-correct clones (fidelity) based on the number of full-length clones.

What are Strings DNA Fragments?

GeneArt Strings DNA Fragments are custom-made, uncloned, double-stranded linear DNA fragments, available in lengths from 150 base pairs (bp) to 3,000, assembled from synthetic oligonucleotides using the same process developed for GeneArt high-quality gene synthesis. Strings Fragments can be used to quickly clone your gene of interest. At least 200 ng of Strings DNA Fragments are produced within 5 (up to 1,000 bp) or 8 (from 1,000 to 3,000 bp) business days. GeneArt Strings DNA Fragments can be designed, sequence optimized, and ordered online and are a cost-effective and smart alternative to clone your expression plasmid.

Are Strings DNA Fragments available below 150 bp?

While we used to offer Strings DNA Fragments between 100 – 150 bp, this is unfortunately no longer possible.

How are Strings DNA Fragments quality-controlled?

Strings Fragments are amplicons (PCR amplificates) of assembled oligonucleotides; an intermediate product of the gene synthesis production process. This process results in a pool of fragments, and cloning and screening need to be carried out to identify the correct clone. To ensure that correct fragments are present in the pool, Strings Fragments are bulk sequence-controlled before shipment.

How are Strings DNA Fragments delivered?

Strings DNA Fragments are shipped dried, ready for resuspension and cloning. Before using, we recommend that you spin down the tube contents, add some water to the bottom of the tube to get the desired concentration, and let it incubate for at least 1 hour at room temperature (alternatively incubate at 4°C overnight). Subsequently, resuspend carefully and use immediately. For longer storage, resuspended Strings DNA Fragments should be dispensed into aliquots and frozen at –20°C. Please avoid freeze-thaw cycles.

Is the production time of all length categories of Strings DNA Fragments the same?
No. Strings DNA Fragments from 150 to 1,000 bp are produced in 5 business days and Strings DNA Fragments from 1,000 to 3,000 bp are produced in 8 business days. Depending on the nature of the sequence, production time can vary.

Are Strings DNA Fragments always cheaper than gene synthesis?

Yes. Since you need to perform the cloning to obtain your final gene, Strings DNA Fragments prices are always below those for gene synthesis.

Are there any gene editing and gene optimization functions available when ordering Strings DNA Fragments?

Yes. We recommend using the GeneArt web order portal for ordering, where you can use the free gene editing and optimization functions to adjust the sequence to meet your needs. After editing and optimization, please check again that the final sequences of the fragment(s) are exactly as needed for further processing in your lab (e.g., potential homologous overlaps of the fragments or restriction sites and buffer bases needed for cloning).

Do you offer services for subcloning or assembly of Strings DNA Fragments to larger genes?

No, subcloning and assembly services are not available for Strings™ DNA Fragments, as they are designed to offer you the fastest and most affordable way to get access to your genes. If you do not want to clone your gene yourself, we recommend that you order full gene synthesis service.

Do you offer the usage of degenerated oligonucleotides to build gene libraries with Strings DNA Fragments?

Yes, we offer GeneArt Strings DNA Libraries from 200 – 2000 bp, containing up to 3 blocks of degenerate nucleotides. Each block can consist of up to 30 bp using the full IUPAC code of DNA nucleotides. Blocks need to be separated by 30 bp of non-degenerate nucleotides.

Can I use Strings DNA Fragments to assemble larger genes?

Yes, it is possible to directly assemble 2 Strings Fragments without pre-cloning. Thermo Fisher Scientific offers several technologies for seamless assembly, e.g., GeneArt Type IIs Assembly or GeneArt Seamless Cloning and Assembly. Please refer to the application examples section on this page for more information. Depending on the sequence length and the number of subfragments you want to assemble, the screening effort to find a correct clone can be high. We therefore generally recommend pre-cloning the subfragments to limit the screening effort required to find a correct clone after assembly.

Are all sequences producible as Strings DNA Fragments?

No. Strings DNA Fragments are limited to 150 to 3,000 bp. If your sequence is longer, you need to order it as gene synthesis or assemble your gene from two or more Strings DNA Fragments. In addition, the Strings DNA Fragments manufacturing process is streamlined to very quickly and cost-effectively provide you with your gene product. If your sequence is complex, you may receive a message that it cannot be produced as a Strings DNA Fragment due to high complexity. In that case, we recommend modifying your sequence to match the production criteria (given below) or ordering your gene as gene synthesis. In many cases, optimizing your sequence using the portal function enables manufacturing with the Strings Fragments process. If it is still not able to be produced, you can manually edit the sequence to enable production.

Important production criteria are:

•    GC content between 20% to 80% (in peaks, not overall content)
•    No long secondary structures or sequence repetitions
•    No A/T stretches >25 bp nor G/C stretches >20 bp

What do I need to consider when cloning Strings DNA Fragments?

Please refer to the section near the top of the page: "Screening Recommendations & Application Examples".

*Production time is the number of business days required to synthesize GeneArt Strings DNA Fragments in our manufacturing facility. Delivery time is in addition to production time and depends on the destination of the shipment.

The research leading to results regarding GeneArt Strings DNA Libraries has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement nr. 613931.