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View additional product information for GeneArt™ Mutagenesis, <1 kb - FAQs (830105DE)
14 product FAQs found
Genes qualifying for Express cloning must be <4 kb and not complex (optimization of your sequence can reduce complexity).
The following vectors are currently available:
- pcDNA 3.1(+)vector
- pcDNA3.3-TOPO vector
- pcDNA3.4-TOPO vector
- pFastBac1 vector
- pET100/D-TOPO vector
- pET151/D-TOPO vector
- pRSET A vector
- pYes2.1V5-His TOPO vector
You should receive your synthesized gene in the selected expression vector. Please note, no additional copy in a pMx cloning vector will be provided as with traditional GeneArt Cloning Services.
Using an alternative production procedure with pre-prepared expression vectors, our GeneArt custom service team can clone your synthesized genes directly into selected Thermo Fisher Scientific expression plasmids, saving 4-5 business days compared to the standard workflow with cloning into a pMx series vector first. Please visit this page (http://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-plasmid-services/geneart-subcloning-service.html) for the differences between Express cloning and Classical cloning.
The kit and custom services offer different advantages. Please click here (http://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis-product-selection-guide.html) to view a selection guide comparing the technologies, including deliverables and production time, to determine what is best for you.
Yes, you can set up mutagenesis bundles as gene variants to order multiple gene variant sequences.
Variants may contain up to four of the following modifications:
- 5'/3' deletion of any length with additional 52 nt of new sequence on each side
- 5'/3' modification of maximum 52 nt on each side
- 5'/3' extension of maximum 52 nt on each side
- Internal modifications of up to 40 nt (a maximum of 3 of these internal modification blocks are allowed)
- Internal deletions of any length
Modifications (whether internal or 5´/3´) have to be separated by at least 100 bp
For GeneArt mutagenesis services, we will need the DNA or amino acid sequence of the gene you would like to mutagenize, the host organism you plan to use (this is important for gene optimization if you choose to include it with your request), the restriction sites you need at the 5'/3' ends and/or to avoid internally, and whether or not you want any other added motifs (e.g., Kozak sequence, stop codons, etc.).
Here are some examples:
- Increase or adjust promoter strength or specificity
- Enhance or modulate protein stability
- Modify or combine enzyme properties
- Increase binding affinities of receptors, ligands, and antibodies
- Optimize or alter signal peptide efficiencies
- Destroy protein function while retaining immunogenicity
- Combine and select natural polymorphisms
- Increase protein half life
- Adjust thermal stability
No material is necessary. For library creation, all we need is the sequence file, submitted electronically, and information about the position and nature of the sites you want to randomize. We can provide a quote for your project through our online ordering system (https://www.thermofisher.com/order/geneartgenes/projectmgmt). If needed, you can then relate detailed information about your library request to our production scientists prior to starting the project.
If you require a degenerate library with an unusual design please send an email to geneartsupport@lifetech.com. We will consider any project and in the vast majority of cases will find a solution to fulfill your requirements.
There are a number of reasons:
- Because you already know that certain amino acid substitutions disturb the function of your protein (e.g., cysteines in complementarity determining regions (CDRs)).
- Because the number of possible variants of a protein is astronomically high, exceeding the capacity of even the highest-throughput screening capabilities by many orders of magnitude. The fewer useless mutations, such as those occurring in less important regions of the protein or that cause frame shifts or stop codons, the better your chances of finding a variant that results in the desired phenotype.
- Some screening assays are cost and labor intensive; thus, screening fewer clones saves time and money.
Conventional protocols for degenerated library creation (e.g., error-prone PCR) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically cause recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independent of their proximity.
The answer depends on the specifics of your project. In general, it is advantageous to keep the diversity of a library as low as possible, targeting only the regions of a gene/protein that are likely to be functionally important. The following information can help determine this: crystal structure, conserved motifs, presence of homologs, etc.