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Custom Gene Synthesis

IDT offers a confidential custom gene synthesis service. By ordering genes from IDT, researchers not only save money spent on reagents necessary for construction, cloning, and sequencing but can also save time by outsourcing the manufacturing of hard-to-clone gene sequences which often result in repeated failures. At IDT, all genes are constructed using Ultramers™, the highest fidelity next generation synthesis technology available. Genes arrive in a plasmid cloning vector and are ready for use in a variety of applications.

Sequence information is always secure and confidential at IDT. Non-disclosure agreements are available through IDT’s legal services upon request.


Documentation and Support


ProductPricing
miniGene 25-400 bp$220.00 USD
Gene 401+ bp$0.55 USD

*The time it takes to make a gene is dependent on length, complexity, and vector choice.

*Extra charges may apply for gene segments with added complexity (such as homopolymeric runs, critical hairpin structures or G/C content), which can interfere with assembly and/or sequencing performance. For more information about potential types of complexity, please see the Overview section below.

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Overview of the Gene and miniGENE ordering process

  1. Sequence entry
    1. Customers may enter their desired DNA sequence using the A,C, G, and T bases only. Degenerate (mixed bases) or modified bases are not offered with our gene products. Alternatively, customers may enter their amino acid sequence consisting of one letter codes. To utilize this feature, customers must also indicate the organism or group of organisms to optimize the codon usage.
    2. Codon optimization
      1. Codon usage can be optimized for a variety of organisms based on either a nucleotide sequence or an amino acid sequence. More information about codon optimization is provided below.
    3. Vector Choice
      1. Customers can choose an IDT in-house vector. Information about these vectors including restriction sites and sequence are available on the Vectors tab.
  2. Automated Review
    1. After submitting your order, the gene request will be reviewed for the following characteristics which may interfere with synthesis, assembly, or sequencing performance:
      1. Secondary structure: Hairpins >10 bases and G/C-rich secondary structures
      2. Repeats and homo-polymeric runs: long repeats, >10 bases for G/C, or >30 bases for A/T
      3. Overall GC Content >65% and regional GC content <25%
      4. Restriction site duplications and Dam/Dcm Methylation sites that may interfere with digestion by restriction endonucleases
    2. If the sequence does not pass the screening criteria, you will be contacted by a gene services specialist to determine the best way to proceed.
  3. Biosecurity review: Each gene will be screened to identify regulated and other potentially dangerous pathogen sequences and must be accompanied by a hazard disclosure statement from the customer. IDT reserves the right to refuse any order that does not pass this screen.

 

 

Codon Optimization

Different organisms vary in the frequency with which they use certain codons. This codon bias is thought to be reflective of the abundance of the corresponding tRNA pool. The fastest growing microbes tend to have the highest codon biases and highly expressed genes tend to closely match the organisms codon bias. Gene sequences containing preferred codons for the expression host are translated more efficiently, with greater accuracy, and in greater yield. In addition, optimization can be used to disrupt difficult sequence features (such GC content, hairpins, repeats, etc.). IDT’s codon optimization tool attempts to match the codon usage to the observed codon bias in the organism’s genes and allows the researcher to review this optimization and minimize sequence characteristics that can interfere with synthesis or sequencing of the gene.

 

Sequence Verification

All Genes and miniGENEs are sequence verified on both strands prior to shipping. Sequence information including chromatograms and a plasmid map, and a FASTA file are available to the customer.

 

Guaranteed yields

IDT will provide 2 µg of purified double-stranded DNA in a circularized plasmid, delivered lyophilized.

 

Confidentiality

All gene information, including sequence entry and choice of parameters, is confidential at IDT.

 

Gene Synthesis Applications

IDT provides the gene in a cloning vector so that it is ready to transform  or further subclone into a vector of the researcher’s choice. These genes can be used in a number of applications including, but not limited to:

  1. Protein Expression of recombinant antibodies, fusion proteins, novel proteins, and functional peptides
  2. microRNA genes
  3. Standards for quantitative PCR and other assays
  4. Template for in vitro transcription (IVT)
  5. shRNA expression cassettes
  6. Regulatory sequence cassettes
  7. Synthetic repeat constructs
  8. Custom vectors
  9. Microarray-ready cDNA
  10. Gene variants and SNPs
  11. DNA vaccines and vectors
  12. Mutation studies, including mutant libraries

      Standard Cloning

      All IDT genes are delivered in a vector ready to be transformed into E. coli. IDT has the ability to construct genes ranging in size from 50bases and larger. Our preferred vector is IDT’s proprietary cloning vector, pUCIDT. Based on the pUC vectors this plasmid is similar to many of the most common plasmids used in molecular biology for the past 3 decades and is available with ampicillin resistance. This vector is particularly well suited for genes larger than 1000 basepairs.

      For those scientists looking for a smaller vector we offer  pIDTSmart. This small (approximately 2kb), high copy vector has been specifically engineered to eliminate the most common restriction endonuclease recognition sites and does not contain a promoter within the cloning region, giving the researcher the flexibility to include the needed sites into the ends of the gene construct. pIDTSmart is available with either Ampicillin or Kanamycin resistance.

      For researchers who desire a more traditional cloning site or who wish to use in vitro transcription, we offer pIDTBlue. Similar to the pBluescript II phagemid vectors, pIDTBlue contains a multiple cloning site in the alpha fragment of the LacZα gene and T3 and T7 promoters flanking the insertion site. This vector is Ampicillin resistant.

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      To Resuspend:

        1. Centrifuge the tube prior to opening to ensure that the DNA is at the bottom of the tube.
        2. Resuspend DNA in 40 µL of IDTE (10 mM Tris, 0.1 mM EDTA) buffer (pH 7.5 – 8.0) to reach an approximate concentration of 50 ng/µL for a stock concentration. Vortex for 20 seconds
        3. Incubate the tube at room temperature for 30 minutes
        4. Centrifuge for 1 minute
        5. To dilute for transformation, add 2 µL of the stock concentration to 999 µL of water to reach an approximate concentration of 0.1 ng/µL. Use 1-2 µL of this working dilution for each transformation

      Recommended Storage:

      • Store hydrated DNA at -20°C.
      • Lyophilized DNA is stable at room temperature for approximately 3 months. We recommend storing at -20°C for longer periods of time.

      Resuspension and Storage Conditions


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      Some common techniques used with our plasmids are listed below. For a more complete description of these and other methods please refer the  following publication:
      Sambrook, Joseph. and Russell, David W. and Cold Spring Harbor Laboratory.  Molecular cloning : a laboratory manual, Joseph Sambrook, David W. Russell  Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. :  2001Transformation (Chemical)

       

      Transformation protocols vary based on the manufacturer and the cell line. For best results we recommend you follow a protocol optimized for the particular cells you are using. Below is a general protocol applicable for many commercial E. coli cell lines such as DH5α or XL1 Blue.

       Place frozen competent cells and pre-labeled tubes on ice

      1. Pre-warm the water bath to 42°C
      2. Once cells are thawed on ice, gently aliquot 25 µL of XL1 Blue cells (Stratagene) to each of the pre-chilled tubes. Keep all the tubes on ice
      3. Add 2 µL of the diluted plasmid (0.4ng) to the cells and gently swirl the cells with pipette tip
      4. Incubate on ice for 30 minutes
      5. Heat shock tubes for 45 seconds in 42°C hot bath
      6. Return tubes to ice for 2 minutes
      7. Add 250 µL of SOC media (recipe below) to each tube and place tubes in a 37°C shaking incubator for 1 hour
      8. Pre-warm agar plates to 37°C in incubator
      9. Spread 125 µL of cells on agar plates containing the appropriate antibiotic
      10. After spreading cells, let plates sit for approximately 10 minutes at room temperature. Then place the plates inverted in 37°C incubator for 12 – 24 hour 

       

      Inoculation and plasmid isolation

       

      1. For a large number of colonies, use a 2.2 mL deep well plate and add 1.6 mL of LB Broth with the appropriate antibiotic. For a small number of colonies, use a 14 mL round bottom culture tube and add 2 mL of LB Broth with the appropriate antibiotic
      2. Touch the colony with a toothpick or pipette tip and place it in the broth
      3. Cover with a gas permeable lid and place in a 37°C shaking incubator for 12 – 20 hours.
      4. Plasmid DNA can be purified from broth cultures by a variety of commercial kits or by the alkaline lysis method listed in the Molecular Cloning  manual by Sanbrook et al. listed above

      Restriction enzyme Digestions

      The standard in-house digestion protocol is below:

       

       

      Incubate for 1 hour at the temperature recommended by the enzyme manufacturer. Take 2-5 µL of the digested sample, add loading dye, and run on a gel to verify that the digestion took place. In separate lanes, also run a ladder to verify the size of the product and a sample of undigested product as a control.

      *The length of running time for the gel will depend on the size of the digested insert. Smaller inserts require shorter run times or the product will run off the gel.

      *The enzyme should not be more than 1/10th of total reaction

       

      DNA 400ng
      Buffer 5µL
      Water XµL
      Enzyme(s) 1µL each
      Total Volume 50µL

      Molecular Biology


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      Material Components Amount (g or ml)/L
      Agar Plates with Amp 1 % Tryptone 10
      1 % Sodium Chloride 10
      0.5 % Yeast Extract 5
      1.5 % Agar 15
      100 µg/ml Ampicillin 0.1
      LB Broth with Amp 1 % Tryptone 10
      1 % Sodium Chloride 10
      0.5 % Yeast Extract 5
      100 µg/ml Ampicillin 0.1
      Agar Plates with Kan 1 % Tryptone 10
      1 % Sodium Chloride 10
      0.5 % Yeast Extract 5
      1.5 % Agar 15
      50 µg/ml Kanamycin 0.05
      LB Broth with Kan 1 % Tryptone 10
      1 % Sodium Chloride 10
      0.5 % Yeast Extract 5
      50 µg/ml Kanamycin 0.05
      SOC Media pH 7.5 2 % Tryptone 20
      0.5 % Yeast Extract 5
      0.05 % Sodium Chloride 0.5
      2.5 mM Potassium Chloride 0.19
      10 mM Magnesium Chloride (hexahydrate) 20.3

      Recipes


      Gene Biosecurity

      Integrated DNA Technologies screens the sequence of every gene order that is received to identify regulated and other potentially dangerous pathogen sequences, and to verify that IDT’s gene customers are legitimate scientists engaged in beneficial research.

      IDT is among the five founding members of the International Gene Synthesis Consortium (IGSC), and helped to create the IGSC’s Harmonized Screening Protocol. The Harmonized Screening Protocol describes the gene sequence and customer screening practices that IGSC member companies employ to prevent the misuse of synthetic genes. IDT takes the steps set out in the Harmonized Screening Protocol to screen the sequences of ordered genes and the prospective customers who submit those orders.

      For more information about the IGSC and the Harmonized Screening Protocol, please visit the website at http://www.genesynthesisconsortium.org/Home.html.

      In October of 2010, the United States government issued final Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic genes should perform gene sequence and customer screening. IDT and the other IGSC member companies supported the adoption of the Screening Framework Guidance, and IDT follows that Guidance in its application of the Harmonized Screening Protocol. For more information, please see 75 FR 62820 (Oct. 13, 2010), or http://federalregister.gov/a/2010-25728.