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Frequently Asked Questions

Gene Synthesis

Ordering from SGI-DNA is faster and less expensive than traditional cloning, saving you time and money. SGI-DNA’s proprietary technology has overcome the challenges of synthesizing large and complex constructs, including genomes, pathways, zinc fingers nucleases, and CRISPR’s. We have successfully synthesized genes with high/low GC content, homopolymers, palindromic structures, and tandem repeats. SGI-DNA leverages the Gibson Assembly Method and intelligent gene design to make large library formats an affordable option to random mutagenesis, while keeping screening loads down.
Fill out this online form and we will send you information and instructions on setting up your account.

You need to submit your sequence in FASTA format and choose one of our standard pUC or BAC vectors. If you would like to use your own vector, you will submit your custom vector’s sequence, including the 5’ and 3’ 100bp overlapping ends.

We offer synthesis of DNA constructs from 125 bp to 2Mbp.

SGI-DNA offers custom gene synthesis, cloning into our standard vector, 500ng of dry DNA, and a quality assurance certificate. Sequencing files are available upon request.

Other services include codon optimization, library design, and plasmid prep to increase yields. See the Plasmid Prep Services page for more details.

Yes, please see our Custom Cloning page for more info. You will need to submit your custom vector sequence and send us your vector. We also require the 100bp 5’ and 3’ sequence overlaps in your vector. Due to unknowns when working with in custom vectors (copy number, toxicity, mutation, etc.), SGI-DNA does not guarantee sub-cloning into custom vectors. After 3 attempts to sub-clone product of interest into customer vector SGI-DNA maintains the right to deliver product of interest in standard SGI-DNA vector of choice International Gene Synthesis Consortium logo

Yes, we offer digitally designed gene libraries. Please see DNA Libraries page. We offer random mutation libraries as well as alanine scanning, antibody, combinatorial site, modular, single site substitution, and truncation libraries.

Gibson Assembly® HiFi 1-Step Kit

  • The Gibson Assembly® Method enables one-step assembly of small and large DNA constructs, using overlapping oligonucleotides or dsDNA fragments as the starting material.
  • Because it is not dependent on restriction enzyme sites, this method can be used to insert DNA fragments into any position of a linearized (restriction-digested or PCR-amplified) vector.
  • The Gibson Assembly® Method is a seamless method leaving no problematic scars at the junctions in your DNA fragment.
  • The Gibson Assembly® Method is much faster than traditional cloning methods.
  • Resulting DNA product may be used immediately for transformation, PCR, or RCA.
  • The Gibson Assembly® Method may be used to construct genes, genetic pathways, as well as genomes, and has the capability of cloning multiple inserts into a vector simultaneously.
  • Can be used to perform site-directed mutagenesis to make simultaneous DNA sequence changes including insertions, deletions, and substitutions.
  • Multiple DNA fragments can be assembled simultaneously in a single reaction.
The Gibson Assembly® HiFi 1-Step Master Mix has been used to assemble DNA fragments greater than 1Mbp. Assembled products as large as 300 kb DNA fragments have been successfully transformed into E. coli, which is the approximate upper limit for cloning into E. coli.
The number of fragments that may be assembled at once is dependent on the length and the sequence of the DNA fragments. We recommend assembling five or fewer inserts into a vector at once in order to reliably produce a clone with the correct insert. For assembly using >5 fragments, we recommend using a strategy involving two or more stages of assembly.
Yes. Design DNA fragments that incorporate the repetitive sequences internally (not at the overhanging ends). This strategy will ensure that each DNA fragment has a unique overlap and will be assembled in the correct order. The repetitive sequence can also be internalized in the first stage of a two-stage assembly strategy. If having the repetitive sequence at the ends is unavoidable, the correct DNA molecules may still be produced at lower efficiencies. Alternatively, longer overlaps that increase the uniqueness of the fragments being joined can be used.
Yes. For optimal results, use these fragments in ≥ 5-fold molar excess.
We have used overlaps as short as 12 bp. As a starting point, we recommend using 40 bp overlaps when assembling dsDNA and 20 bp overlaps when assembling ssDNA oligonucleotides.
To significantly reduce the background of unwanted vector-only colonies, use PCR-derived vector rather than a restriction fragment vector. If background continues to be a problem, purify the PCR-amplified vector from an agarose gel following electrophoresis.
Yes. Because a covalently joined DNA molecule is produced, it may be PCR-amplified. In addition, if the final product is a circle, it may be used in rolling-circle amplification reactions with φ29 polymerase.
The kit is optimized for the assembly of DNA molecules with ≤ 100 bp overlaps.
Yes. However, the optimal concentration of each oligonucleotide should be empirically determined. As a starting point, use 45 nM of each oligonucleotide. Keep in mind that oligonucleotides > 90 bases may have secondary structures that interfere with assembly.
Yes. The assembly reaction has been optimized for a 1 hour, 50°C incubation. However, extended incubation times (e.g. 2–16 hours) have been shown to improve cloning efficiencies in some cases in many cases. Alternatively, for the assembly of ≤3 fragments, 15 minutes has been shown to be sufficient. Reaction times less than 15 minutes are not recommended.
The reaction has been optimized for DNA assembly at 50°C but has been demonstrated to work between 40°C and 50°C.
Restriction enzyme inactivation is only necessary if the insert contains the restriction site recognized by the restriction enzyme used for vector linearization.
This is generally not necessary. A cleanup kit or a standard phenol-chloroform extraction followed by ethanol precipitation is sufficient. We have also directly used unpurified PCR products in assembly reactions.
We have successfully used this method in combination with a variety of chemically competent and electrocompetent E. coli cells. However, TransforMax™ EPI300™ Electrocompetent E. coli consistently produce the greatest number of clones and the highest efficiency of the correct insert.
  • Assemble and transform the positive control provided in the kit. The successful cloning of the positive control will demonstrate that the assembly mixture is functional and the transformation conditions are suitable.
  • Analyze the reaction on an agarose gel. An efficient assembly reaction will show assembled products of the correct size and the disappearance of the starting substrate DNA fragments.
  • Check the design of the overlapping DNA fragments.
  • Consider whether the cloned insert may be toxic to E. coli and a whether a low-copy vector, such as a BAC, should be used.
  • Because the assembled product is a covalently closed molecule, it may be PCR-amplified. Consider whether this is suitable for your purposes.

Gibson Assembly® Ultra Kit

  • The Gibson Assembly® Method enables one-step assembly of small and large DNA constructs, using overlapping oligonucleotides or dsDNA fragments as the starting material.
  • Because it is not dependent on restriction enzyme sites, this method can be used to insert DNA fragments into any position of a linearized (restriction-digested or PCR-amplified) vector.
  • The Gibson Assembly® Method is a seamless method leaving no problematic scars at the junctions in your DNA fragment.
  • The Gibson Assembly® Method is much faster than traditional cloning methods.
  • Resulting DNA product may be used immediately for transformation, PCR, or RCA.
  • The Gibson Assembly® Method may be used to construct genes, genetic pathways, as well as genomes, and has the capability of cloning multiple inserts into a vector simultaneously.
  • Can be used to perform site-directed mutagenesis to make simultaneous DNA sequence changes including insertions, deletions, and substitutions.
  • Multiple DNA fragments can be assembled simultaneously in a single reaction.

BioXp™ 3200

BioXp™ system DNA fragments are assembled using a high fidelity DNA polymerase that result in blunt ends. These ends are compatable with most homologous recombination methods such as Gibson Assembly® cloning, traditional ligation, and TOPO® blunt end cloning. BioXp fragments have been validated using homologous ends using Gibson Assembly® cloning.
GA ends are 30 nucleotide flank sequences that allow for Gibson Assembly® of BioXp™ fragments. GA ends allow BioXp™ fragments to be readily cloned into compatible vectors using the Gibson Assembly® HiFi 1-Step or Ultra cloning reagents.
GA ends are designed for use in Gibson Assembly® cloning methods, so that BioXp™ fragments can be cloned directly into the pUCGA1.0 vector. BioXP™ fragments can also be used in most cloning, restriction digest, and PCR amplification reactions. GA ends can be removed by restriction digest or PCR amplification, when necessary. For questions about specific applications, please contact technical services.
De novo DNA synthesis is sequence dependent. Repetitive sequences of high GC content may cause poor to low DNA synthesis. If the synthetic DNA is weak or has multiple bands, it's recommended to gel purify the correct size band from the product before continuing with the workflow.
There are a few of possibilities: 1) The fragment failed to assemble. 2) The fragment is smaller than 400bp. Smaller fragments will yield little to no fragments during purification. 3) Poor recovery was obtained during the error correction step. Please contact technical support for additional assistance.
The BioXp™ system also requires 50 ul and 200 ul tips which are sold separately. Amounts required depend on the total number of assembly reactions purchased. We recommend you load 3 x 50 ul tips and 1 x 200 ul tips with the start of a new run. You can save any unused tips for another time.
In addition to the BioXp™ kits provided for DNA assembly, fresh 70% ethanol solution must also be added to the system reagent reservior before starting the protocol.
The BioXp™ system provides linear DNA fragments with blunt ends in TE buffer. Additionally, GA ends (30bp at 5' end and 30bp at 3' end) were added to assembly input sequences. BioXp™ fragments with GA ends can be easily cloned into a Gibson-ready subcloning vector. We recommend using the Gibson Assembly® Ultra Kit to clone your BioXp™ fragment into the pUCGA1.0 vector (sold separately).
BioXp™ compatible constructs fall within a 400-1800 bp range and are 40 - 60% GC rich. At the time of construct submission, sequences are analyzed for compatibility. Some gene structures increase the complexity of assembly reactions and are therefore deemed incompatable. If sequences fall out of the BioXP™ specifications, you will be provided with a quote for our in-house custom DNA synthesis. For questions about compatibility, please contact technical services.
To place an order, log on to the BioXp™ ordering portal using your account information, complete the required information, and submit a fasta file with your constructs. The system will generate an estimated cost for all constructs within the BioXp™ parameters. Any constructs outside of the complexity analysis or flagged as a potential dangerous pathogen will result in the need for additional information.
BioXp™ constructs are analyzed for both complexity and biosecurity. Any constructs falling outside of the complexity analysis will be removed from the BioXp™ order and offered as an optional custom synthesis order. Any construct flagged as a potential dangerous pathogen will result in the need for additional information from the customer.
A 1.8 kb DNA Assembly Reagent kit includes: (1) Oligo Vault Pool, (1) 96 well 1.8 kb DNA Assembly Reagent Plate, (1) EC Strip, (1) DNA Purification Strip, and (1) Recovery Plate. Fresh 70% ethanol, 50 ul tips, and 200 ul tips must be provided separately.
The BioXp™ system is capable of producing constructs ranging from 400-1800 bp.
There are many facets to consider when assembling linear DNA fragments. We anticipate ≥85% of constructs within the defined BioXp parameters will be successfully assembled by the BioXp™.
Yes, SGI-DNA has both high copy number and low copy number vectors available for cloning BioXp™ products.
The final product is in 45µl TE buffer. The concentration is construct dependent. In general, we see 200-800ng.
Any run interrupted once the protocol has been initiated, may be unrecoverable. There is a chance that you can retrieve a sufficient intermediate product for your experiments. If this occurs, please contact technical services for more direction.
The BioXp™ system requires an ethernet port with access to the internet. This allows instrument protocols to be delivered to the system and remote troubleshooting availability. For more information, please review the setup and installation guide.
The BioXp™ system is able to process 32 constructs per run.
DNA Assembly of 1.8 kb takes approximately 17 hours to run.
An interruption of power will abort the current job. We highly recommend using an uninterruptable back up power supply (UPS) of ~1000 watts and 1500KVA or more.
TheBioXp™ system capabilities range from 400 bp to 1800 bp and 40% - 60% GC rich with minimal complexity. An analysis will be completed upon sequence submission at the time of order.
Yes, you can cancel a job during the delay start. You will need new reagents if you choose to begin the run another time. The Oligo Vault and components contained within the +4C box can be returned to +4C until use. If you would like to restart the job immediately, please contact tech services to walk you through a possible option.

Trademark Information

Synthetic Genomics® and Gibson Assembly® are registered trademarks of Synthetic Genomics Inc.
TransformMax™ and EPI300™ are trademarks of Epicentre Technologies Corporation.
Gibson Assembly® : US Patent Nos. 7,776,532 and 8,435,736
© 2014 Synthetic Genomics Inc. All rights reserved.