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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.

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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.

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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.

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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.

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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

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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.).

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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.

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It is important to differentiate naturally occurring mutations linked to the chemical nature of the oligo manufacturing process from the perceived mutations that occur when desalted oligos are used in certain applications.

If the mutation you find is not consistent in all of your clones, this probably falls into the category of a "perceived" mutation that is actually an expected by-product of an oligo reaction. This has a few causes:

-Following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 30-mer was synthesized, the solution would also contain 29-mer failures, 28-mer failures, 27-mer failures etc. The amount of failure sequences present is influenced by the coupling efficiency. For an oligo of this type, the percentage of full-length oligo would be between 74 and 54%, assuming a 99 or 98% coupling efficiency (the percentage of full-length oligos produced from a given synthesis declines as the length of the oligo increases). The truncated oligos usually result because oligos are synthesized from 3' to 5' end. Primers that are desalted and not purified for length will have truncated product missing one or more bases at the 5' end. Hence, oligos that are desalted are only recommended for diagnostic PCR, microarrays, or sequencing. We recommend purification of the oligos if they will be used in certain demanding applications such as mutagenesis or cloning, especially if restriction sites are added to the 5' end of them.

-Other sources of perceived mutations for both desalted and purified oligos are sequencing artifacts, point mutation introduced during PCR, unstable stem loop structures in the primers, propagation of the plasmid DNA after cloning in an E. coli strain that is muS, mutD or mutT or a silent mutation selected by the bacterial strain because of codon usage in that strain.

Naturally occurring mutations are a rare but inherent event in the chemical synthesis of the oligos. The chances of having one single insertion or deletion in a given oligo of about 30 bases is about 2%.

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1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.

2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.

3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).

4. High-throughput Yeast Two-Hybrid Assays for Large-Scale Protein Interaction mapping. Methods: A Companion to Methods in Enzymology 24(3), pp.297-306

5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.

6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.

7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.

8. Protein-over expression and crystallography Evdokimov, A.G., Anderson, D.E., Routzahn, K.M. & Waugh, D.S.

9. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis. Acta Crystallography (2000) D56, 1676-1679.

10. Evdokimov, et al. Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallographica D57, 793-799 (2001).

11. Lao, G. et al. Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells. Cryobiology 43(2):106-113 (2001).

12. High-throughput cloning and expression Albertha J. M. Walhout, Gary F. Temple, Michael A. Brasch, James L. Hartley, Monique A. Lorson, Sander Van Den Huevel, and Marc Vidal.

13. Gateway Recombinational Cloning: Application to the Cloning of Large Numbers of Open Reading Frames or ORFeomes. Methods in Enzymology, Vol. 328, 575-592.

14. Wiemann, S. et.al., Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs, Genome Research (March 2001) Vol. 11, Issue 3, pp.422-435

15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination

16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)

17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs

18. Loftus S K et al. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 31;8(5):221 (2001).

19. James L. Hartley, Gary F. Temple and Michael A. Brasch. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research (2000) 10(11), pp. 1788-1795.

20. Reboul et al. Open-reading frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nature Genetics 27(3):332-226 (2001).

21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).

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Combinatorial Libraries (up to 1,011 variants): Simultaneous randomization of multiple codons, TRIM technology optional
Site-Saturation Mutagenesis (up to 20 variants): Randomization of a single codon with every possible non–wild type variant
Sequential Permutation Libraries (# of codons x 20 variants): Successive site-saturation mutagenesis
Controlled Randomization (up to 1,011 variants): Unbiased random substitutions with defined frequency
Cloned cDNA of all known human SH3 domains
Truncation Libraries: Customer-defined truncations without out-of-frame mutations.
GeneArt Strings DNA Libraries: linear DNA fragments that can contain up to three regions of randomized nucleotides

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The Template Generation System Kit provides a method for introducing primer binding sites randomly into foreign target DNA. Insertion of the Entranceposon into foreign DNA makes it possible to sequence the flanking DNA using the primers supplied in the kit. The clones for targeted sequencing can be selected from the population of random insertions by using simple colony-PCR or restriction enzyme mapping.

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The differences are as follows:

- The GeneArt kit combines the methylation and DNA amplification steps, saving 1 hour in the protocol.
- The enzyme mix from the GeneArt Seamless Cloning and Assembly Kit is part of the protocol, boosting the colony output without the need to do many PCR cycles (another time saving step).
- The new kit also contains an enhancer used during the PCR step that increases mutagenesis efficiency for a wide range of plasmid sizes.

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Before you can transfect your expression clone into 293A cells, you must expose the left and right viral inverted terminal repeats (ITRs) on the vector to allow proper viral replication and packaging. This also removes bacterial sequences (i.e., pUC origin and ampicillin resistance gene). Both pAd/CMV/V5-DEST and pAd/PL-DEST ;vectors contain Pac I restriction sites (see maps on pages 20 and 22 of the manual (http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf), respectively, for the location of the Pac I sites).

Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. If you are unable to use the Pac I site, you can use the Swa I site.

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These guidelines may be useful as you design your PCR primers:

- In general, a length of 18-30 nucleotides for primers is good.
- Try to make the melting temperature (Tm) of the primers between 65 degrees C and 75 degrees C, and within 5 degrees C of each other.
- If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
- Typically, 3 to 4 nucleotides are added 5' of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in TOPO cloning, the primers should not have a phosphate modification.
Read more about primer design tips and tools at https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.

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Invitrogen Custom DNA Primers are offered with several options for primer purification. Which one you should choose depends on your application. Here are some details on the options that are typically available:

Desalted--this is our standard purification procedure. The primer will be gas-phase deprotected and normal-phase desalted. Organic products are removed with 10% acetonitrile/90% water wash, and the products are then eluted under aqueous conditions. The smallest oligo we synthesize at this purification is 5 bases. Desalting removes salt, but not failure sequences. This method is sufficient for standard PCR, RT-PCR, cDNA synthesis, and sequencing reactions.

Cartridge Purification--this is based on reversed-phase chromatography to provide full-length sequences. The majority of the n-1, n-2, n-3, etc. species will be removed for oligos less than 35 bases. The smallest oligo we synthesize at this purification is 7 bases. Cartridge Purification is recommended for PCR with primers that have critical 5' sequences (RE sites, promoters), site-directed mutagenesis, first-strand cDNA synthesis for generation of libraries, gel shift assays, and production of cloning adapters.

HPLC (high-pressure liquid chromatography)--HPLC is a reversed-phase chromatography method similar to that used in cartridge purification. This process removes failure sequences and unincorporated labels. This is offered for oligos up to 50 bases long. HPLC is recommended for antisense work.

PAGE Purification--a denaturing polyacrylamide gel is used to purify the full-length primers from ALL types of n-1, n-2, etc. species of oligos. Oligos are purified by electroelution from the acrylamide gel and then run on a desalting column. These primers are not phenol extracted. PAGE purification can be used for any length of oligonucleotides. Typical yields for PAGE purified primers are not as high as when purified by other methods, but the product is highly pure. This is recommended for GeneTrapper screening. For more information, refer to the Oligos pages under Products & Services on our website.

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