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. 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.

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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GeneArt Strings compared to gBlocks White paper

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

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

Figure 1. 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., Invitrogen 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. 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.

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.

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.

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.

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 colonies were sequenced to determine the number of sequence-correct 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.

*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.