New Easy Methods for Next Generation Cloning for Every Lab Webinar Q&A

Thank you for joining our Next Generation Cloning webinars. Here are the questions and answers collected from attendees of the live webinar sessions:

Find the answer quickly by clicking your desired question.

Q:  In one sentence, what are GeneArt Strings DNA Fragments?
Q:  If the potential DNA fragment contains secondary structure, is the final quality of the recombined plasmid affected?
Q:  Do you offer a service for randomizing specific residues of the DNA, i.e. for directed molecular evolution?
Q:  Can one incorporate chemically modified nucleotides, such as etheno-dCTP or methyl-dCTP, during the GeneArt Strings DNA Fragments synthesis?
Q:  Is this method suitable for degenerate sequences (i.e., for randomized codon positions)?
Q:  What method would you advise to assemble a 20-kb fragment?
Q:  Why do you have a 1 kb limit on Strings?
Q:  What is the maximum gene length that I can clone with this technique?
Q:  Do you send single-stranded or double-stranded fragments?
Q:  Is double-stranded linear DNA suitable for cell line transfection?
Q:  Say one vector is too big to transfect, could you transfect it with two of these vectors and have the recombination occur inside the cell by expressing the recombinase on one vector?
Q:  What is the approximate cost for a 1 kb gene fragment?
Q:  How much will it cost for a 3 kb gene?
Q:  I need to clone a putative protein but I don't know the number of introns. How can I resolve this problem for cloning?
Q:  What is the status of the 5' and 3' ends of the DNA Strings? Is this compatible with A addition for Topo-TA cloning?
Q:  The design tool that designs primers for recombination-based assembly of fragments: can you vary the length of the homology?
Q:  With regards to the 15 bp homology question: I typically use yeast recombination to assemble my fragments. A design tool that would allow me to design 30-50 bp of homology would be useful.
Q:  What is the error rate associated with Strings production? How many mutations would I expect to see in any given construct?
Q:  Are all Strings sequence validated?
Q:  How does Life Technologies prevent the ordering of DNA that could potentially be dangerous (such as the synthesis of DNA that encodes toxins, viral components, etc.)?
Q:  Is Seamless cloning the same as Gibson assembly?
Q:  Do you recommend to optimize codons for mammal genes for better protein expression even in mammal expression systems?
Q:  If the sequence is not 100% correct, is it usually incorrect at the ends or in the middle of the fragment?
Q:  Why is it that ordering Gene Synthesis avoids problems with sequence complexity that cannot be avoided with GeneArt Strings?
Q:  What is the difference between GeneArt Strings Fragments service and Gene Synthesis?
Q:  What about genes from little-studied organisms? How can these genes can be synthesized without prior knowledge of their gene sequences through traditional cloning?
Q:  Do you have codon optimization for filamentous fungi? Aspergillus, neurospora, etc.
Q:  How should we deal with Strings requests that come back as too complex if we do not have the freedom to optimize (i.e. we require a specific DNA sequence)?
Q:  Is there a way to account for optimization deviation? Say a gene is expressed at a lower level intentionally and has a strategic GC content that is not necessarily optimal for certain domains or sequences that is used as part of the regulation in a circuit? Can you add a level of deviation from the optimized code?
Q:  Will you be offering larger fragment sizes such as 1.8 kb?
Q:  What do I do when I need more than 200 ng of DNA fragment?
Q:  How does the GeneArt cloning system differ from Clonetech's InFusion?

Q: In one sentence, what are GeneArt Strings DNA Fragments?

A: Strings fragments are affordable custom-made, linear, double-stranded DNA fragments up to 1 kb in length, ready for cloning in your lab.

Q: If the potential DNA fragment contains secondary structure, is the final quality of the recombined plasmid affected?

A: Secondary structures are in general an issue for efficient cloning and sequencing of the affected region, as well as for Strings DNA Fragments production. If you use our online portal for ordering you might receive a notification that the sequence cannot be produced as a Strings DNA Fragment due to sequence complexity (i.e. secondary structures) and sequence optimization might solve the problem. If the sequence is still too complex after optimization, we recommend you order as Gene Synthesis. The quality of the plasmids are not affected.

Q: Do you offer a service for randomizing specific residues of the DNA, i.e. for directed molecular evolution?

A: Yes- we offer Directed Evolution services. Please see GeneArt® Controlled Randomization Service and GeneArt® Combinatorial Libraries Featuring TRIM Technology (/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis/directed-evolution.html). However, Strings fragments can only be ordered with the four standard nucleotides (bases other than G, A, C, T are not offered).

Q: Can one incorporate chemically modified nucleotides, such as etheno-dCTP or methyl-dCTP, during the GeneArt Strings DNA Fragments synthesis?

A: No- Strings can only be ordered with the standard, unmodified nucleotides G, A, T, C.

Q: Is this method suitable for degenerate sequences (i.e., for randomized codon positions)?

A: No- Strings fragments can only be ordered with G, A, T, C nucleotides, without modifications.

Q: What method would you advise to assemble a 20-kb fragment?

A: The GeneArt Seamless PLUS Cloning and Assembly Kit (A14603) can be used to assemble up to 4 fragments and create constructs up to 40 kb.

Q: Why do you have a 1 kb limit on Strings?

A: Strings fragments are limited to 1000 bp due to the very streamlined production process to enable the fastest delivery time and price.

Q: What is the maximum gene length that I can clone with this technique?

A: For direct assembly we recommend to join only two Strings DNA Fragments together to limit your screening effort. For larger genes we suggest to pre-clone and sequence verify the fragments before further assembly. This will help limiting your screening effort. The maximum length will be determined by your cloning method of choice. For example, there is no upper limit for Gateway cloning, whereas GeneArt® Seamless PLUS Cloning & Assembly Kits accommodate four fragments up to 10 kb each.

Q: Do you send single-stranded or double-stranded fragments?

A: Strings are blunt double-stranded DNA fragments.

Q: Is double-stranded linear DNA suitable for cell line transfection?

A: Yes- in principle, it is suitable for transfection. However, we deliver a pool of DNA fragments that has limited copies of full-length and completely correct fragments. So if you use Strings for transfection you might end up with low numbers of cells transfected with the correct DNA.

Q: Say one vector is too big to transfect, could you transfect it with two of these vectors and have the recombination occur inside the cell by expressing the recombinase on one vector?

A: For very large constructs we recommend you work in yeast and use the GeneArt High-Order Genetic Assembly System (A13285); this can be used for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 kb in length, into any vector.

Q: What is the approximate cost for a 1 kb gene fragment?

A: Strings come in 3 categories: 100 to 600 bp for $99; 601 to 750 bp for $129; 751 to 1000 bp for $149 USD.

Q: How much will it cost for a 3 kb gene?

A: You can assemble a 3 kb gene yourself from three 1 kb Strings ($149 for 1 kb) using your choice of assembly technique (i.e. GeneArt® Seamless PLUS Cloning & Assembly Kit).

Q: I need to clone a putative protein but I don't know the number of introns. How can I resolve this problem for cloning?

A: We recommend to find out the full sequence before ordering.

Q: What is the status of the 5' and 3' ends of the DNA Strings? Is this compatible with A addition for Topo-TA cloning?

A: There are neither end phosphates nor A overhangs resulting from the Strings Fragments production process. This is compatible with TOPO cloning, as phosphates are inhibitory. For TA cloning, one can perform a PCR tailing reaction to add the A overhang.

Q: The design tool that designs primers for recombination-based assembly of fragments: can you vary the length of the homology?

A: Strings DNA fragments are suitable for any recombination-based method. We recommend to adapt the length of the homologous region to the method of your choice (i.e. 15 bp homology for GeneArt® Seamless PLUS Cloning & Assembly Kits).

Q: With regards to the 15 bp homology question: I typically use yeast recombination to assemble my fragments. A design tool that would allow me to design 30-50 bp of homology would be useful.

A: Unfortunately, the length of homologous regions cannot be varied using the GeneArt Seamless Cloning & Assembly web design tool. We suggest to design your homologous regions and verify using a sequence alignment program.

Q: What is the error rate associated with Strings production? How many mutations would I expect to see in any given construct?

A: The probability of finding a correct clone is >90% when one sequences 4-8 full-length clones.

Q: Are all Strings sequence validated?

A: Bulk sequencing is a part of the quality control of Strings DNA Fragments to verify your sequence is present in the pool.

Q: How does Life Technologies prevent the ordering of DNA that could potentially be dangerous (such as the synthesis of DNA that encodes toxins, viral components, etc.)?

A: An internal Biosafety and Biosecurity check is performed with every Strings sequence, as for all Gene Synthesis projects. If a match is found in our database for indexed sequences, the customer is informed that we need a BAFA application at the German authorities to determine if the sequence can be produced. It may take several weeks until the application is granted. If it is denied, the order will be refused.

Q: Is Seamless cloning the same as Gibson assembly?

No- Seamless Cloning is a technology based on homologous recombination. It is different from Gibson Assembly.

Q: Do you recommend to optimize codons for mammal genes for better protein expression even in mammal expression systems?

A: Yes- optimization is also recommended for autologous expression; see Fath S, Bauer AP, Liss M, Spriestersbach A, Maertens B, et al. (2011) Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression. PLoS ONE 6(3): e17596. doi:10.1371/journal.pone.0017596

Q: If the sequence is not 100% correct, is it usually incorrect at the ends or in the middle of the fragment?

A: Errors can be present throughout the sequence.

Q: Why is it that ordering Gene Synthesis avoids problems with sequence complexity that cannot be avoided with GeneArt Strings?

A: The Strings production process has been optimized for speed and low cost to make Strings a quick and affordable alternative to Gene Synthesis. Complex sequences have to be produced with an alternative gene synthesis protocol, which involves several other/additional production steps.

Q: What is the difference between GeneArt Strings Fragments service and Gene Synthesis?

A: The main difference is that Gene Synthesis is not length restricted and comes cloned in a standard plasmid and 100% sequence verified, whereas Strings are linear, double-stranded DNA fragments up to 1,000 bp in length. For more information, please refer to the Strings DNA Fragments webpage

Q: What about genes from little-studied organisms? How can these genes can be synthesized without prior knowledge of their gene sequences through traditional cloning?

A: If the sequence of your gene is not known, we can not produce it as a String.

Q: Do you have codon optimization for filamentous fungi? Aspergillus, neurospora, etc.

A: We offer the same codon usage tables as for Gene Synthesis; if you need special optimization, please contact the support team under GeneArtSupport@lifetech.com.

Q: How should we deal with Strings requests that come back as too complex if we do not have the freedom to optimize (i.e. we require a specific DNA sequence)?

A: Please order as regular Gene Synthesis. Strings production process has been optimized for speed and low cost, whereas we perform several additional production steps during Gene Synthesis.

Q: Is there a way to account for optimization deviation? Say a gene is expressed at a lower level intentionally and has a strategic GC content that is not necessarily optimal for certain domains or sequences that is used as part of the regulation in a circuit? Can you add a level of deviation from the optimized code?

A: No- we do not offer special optimization for Strings fragments. Please order your sequence as Gene Synthesis and contact GeneArtSupport@lifetech.com for special optimization, or simply order your sequence as wild type.

Q: Will you be offering larger fragment sizes such as 1.8 kb?

A: Yes- at sometime in the future.

Q: What do I do when I need more than 200 ng of DNA fragment?

A: We guarantee at least 200 ng, but we usually deliver between 500 and 1000 ng. You can also use the sequences of amplification primers we provide on every Strings documentation sheet to order oligos and re-amplify your Strings.

Q: How does the GeneArt cloning system differ from Clonetech's InFusion?

A: GeneArt Strings DNA Fragments are delivered as dried double-stranded synthetic DNA ready for cloning, and no additional cloning reagents are delivered. The GeneArt Seamless Cloning & Assembly technology is based on homologous recombination and is suitable for Strings assembly.