TechVault
  • What types of sequence motifs should be avoided when ordering gBlocks® Gene Fragments?

    Sequences that cause particular problems for gBlocks Gene Fragment synthesis are those having extremely low or high GC content (less than 25% and greater than 75%), homopolymeric runs of 10 or more As and Ts or 6 or more Gs and Cs. Other structural motifs such as repeats or hairpins may also influence our ability to construct gBlocks Gene Fragments. The decision to accept or reject an order for a particular gBlocks fragment sequence is complex and multifactorial. All of our gBlocks fragments are reviewed by our Gene Specialists prior to manufacturing to ensure the sequences can be made successfully. Questions about gBlocks Gene Fragments? Contact our Gene Specialists directly at genes@idtdna.com.

  • Does IDT have products for Next Gen Sequencing (NGS) that are not platform specific?

    IDT can synthesize custom adaptors, fusion primers, Multiplex Identifier (MID) sequences, and other workflow oligonucleotides for next generation sequencing (NGS). We supply both xGen™ Lockdown™ Probes (biotinylated Ultramer™ probes) and 200 pmole Ultramer DNA Plate Oligos for in-solution target capture to enrich known sequences within a genome. TruGrade™ Processing Service (request info) is also available to improve the accuracy of NGS, which increases bar code binning during post-sequencing analysis by up to 30X compared to competitor oligonucleotides. For more information about IDT NGS products, go to Next Gen Sequencing on the Products tab at www.idtdna.com.

  • What is the maximum size construct that can be assembled with gBlocks® Gene Fragments using Gibson Assembly®?

    The original paper describing the Gibson Assembly® Method [Gibson et al., 2009] suggests the method can be used to assemble constructs in the size range of small bacterial genomes, and megabase assemblies have been demonstrated using this protocol. However, at IDT we have limited our gBlocks fragment assemblies to 2–2.3 kb gene constructions using 4–5 gBlocks Gene Fragments using the Gibson Assembly Master Mix, available from New England BioLabs. For larger assemblies, we recommend a sub-assembly strategy with sequence-verification of the component parts.

    Reference: Gibson DG, Young L, et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6 (5):343–345. doi:10.1038/nmeth.1318. PMID 19363495.

  • Why do the scale I order and the amount of oligo I receive differ—I receive less than the requested scale quantity?

    The scale you order determines the amount of sequence-initiating base used to begin synthesis of your oligonucleotide. Since oligonucleotide synthesis (or any chemical reaction) is never 100% efficient, the amount of final product recovered after synthesis and purification steps (yield) will always be less than the starting amount used for synthesis. Modifications, secondary structures, and purification services further affect the final yield for a given construct. IDT offers guaranteed yields based on all of these factors that define the minimum amount of oligonucleotide you will receive for a given construct.

    The minimum yield guarantee is listed in the shopping cart and in your confirmation email. To receive a higher final yield, select a larger synthesis scale. For further discussion on this topic, see the Technical Report, Oligonculdotide Yield, Resuspension, and Storage.

  • Are you able to synthesize DNA oligos containing deoxyuridine (dU) instead of (deoxythymidine) dT?

    We routinely make oligos that include deoxyuridine in place of deoxythymidine bases. The minimum synthesis scale for oligos containing dU is 100 nmoles. The modification code for internal deoxyuridine in a sequence is /ideoxyU/, while 5’ and 3’ incorporation have the modification codes of /5deoxyU/ and /3deoxyU/, respectively. More details, including pricing information, are provided on the Modifications page under the Products tab at www.idtdna.com.

  • When performing NGS, when is enrichment/target capture of genomic sequences necessary?

    You should consider target enrichment (target capture) if you are analyzing a subsection of the genome or performing deep sequencing to detect rare mutations. This allows you to re­serve sequencing capacity for the region(s) of interest, and reduce the sequencing cost and time associated with data analysis. IDT xGen™ Lockdown™ Probes are useful for enriching genomic regions ≤250 kb. They are also excellent for supplementing other capture panels to expand the target region or improve enrichment performance.

  • For what applications are Ultramer® Oligonucleotides useful?

    Ultramer Oligonucleotides are high fidelity, long, single-stranded oligos of up to 200 bases. They are suitable for demanding applications such as cloning, DNA-directed RNA interference (ddRNAi), and gene construction, and can save researchers a great deal of time and trouble through direct synthesis of the en­tire target fragment. For example, Ultramer Oligonucleotides can be used to efficiently and quickly generate large insertions and deletions, or change stretches of sequence identity. They can also be used as templates for the synthesis of RNA in in vitro transcription or as DNA standards in qPCR.

  • Why can’t I select a quantity when I order custom oligos?

    IDT Custom DNA Oligos are available at several synthesis scales and are usually supplied dried down. Therefore, we do not offer the option for a quantity of tubes. Even with our lowest synthesis scale, the yield delivered is sufficient for thousands of reac­tions with most standard applications. Due to this excess of product, most researchers resuspend oligos as a concentrated stock solution and, from that, create several tubes of working solution. IDT also offers several options for normalization and preparative services. Please contact Customer Care at custcare@idtdna.com for additional informa­tion on custom formulation.

  • What tools does IDT provide to design multiplex qPCR assays?

    Use the free IDT OligoAnalyzer® software (www.idtdna.com/scitools) to ensure that primer and probe sets lack hairpins, and homo- and heterodimer interactions. From there, you can link directly to the NCBI BLAST tool to further analyze possible sequence interactions. For help with using the BLAST tool, see Tips for Using BLAST to Locate PCR Primers, in DECODED 1.1, April 2011. For more information on setting up multiplex qPCR assays, see the article, Multiplex qPCR—How to Get Started, in DECODED 3.2, April 2013.

  • How do I choose the best internal control/reference/housekeeping gene for normalizing qPCR results?

    IDT recommends that you test at least two, but preferably three, normalizing or house­keeping genes to ensure accurate internal controls. The best normalizing gene to use will depend on the species and conditions of the sample you will be testing. If you are unsure of the best normalizing gene to use, review the literature for the genes tested on samples with conditions similar to yours. When normalizing to a reference gene, it is very important that the reference gene is ex­perimentally validated to ensure that it is an accurate measure against which to compare all other sample variations. A pilot study can be conducted to select the best set of ref­erence genes out of a series of candidates. Analysis of reference gene stability can be performed with tools such as geNorm or qbase+ software (http://www.biogazelle.com/). The reference genes should have stable mRNA expression and the amount of reference gene mRNA should be strongly correlated with the total amounts of mRNA in the samples. When using this method, it is critical that the reference genes used do not vary with experimental conditions. Normal­ized data is reported as a ratio of the mRNA concentration of the gene of interest to the mRNA concentration of the reference gene. This can be calculated by a comparative Cq method or a standard curve method. For in depth discussion and recommendations on this topic, see the IDT webinar by Stephen Bustin & IDT, MIQE Guidelines: A Roadmap for Proper qPCR Experimental Design and Reporting,

  • How do I indicate a hand mixed degenerate or random (wobble) base in my sequence when I order?
    “Hand-Mixed Bases”, also known as degenerate, random, or wobble bases, can be introduced at any position in an oligonucleotide sequence. We use a different notation for Hand-Mixed Bases than for our standard machine mixed bases. Thus, when inputting your sequence, you simply choose the mixed base letter (IUB symbol) that represents the desired base mix from the table below.



    For the first occurrence of a particular mixed base, the proportion of each base must be indicated, in the order ACGT. For example, an N mixed base that requires 25% of each included base will be indicated as (N:25252525); if the bases are required in the proportions 26% of A and T, and 24% of C and G, the mixed base will be represented as (N:26242426). Each mixed base after the first insertion of that ratio will not need the ratio, just the base in parentheses, e.g. (N).

    Multiple mixes are possible, for a total of 4 maximum per oligo.
    See example sequences below:

    Example 1: ACGSSSACT with 50% of C and G for all of the S bases would be indicated as follows: ACG(S:00505000)(S)(S)ACT 

    Example 2: ACTGACTGNNNNNNNNNACTGATGC with 25% of A, C, G, and T for all of the N bases would be indicated as follows: ACTGACTG(N:25252525)(N)(N)(N)(N)(N)(N)(N)(N)ACTGATGC

    Example 3: ACTGACTGNNNNNNNNNNCAGTACTG with the first 5 N-bases being equimolar, and the latter 5 N-bases being a 70/10/10/10 mix (favoring A): ACTGACTG(N1:25252525)(N1)(N1)(N1)(N1)(N2:70101010)(N2)(N2)(N2)(N2)CAGTACTG

    Please contact IDT Customer Care at custcare@idtdna.com with any further questions. 

  • How can I check my PCR primers using the OligoAnalyzer® program to ensure there are no significant primer design issues?
    Look for PCR primers that conform to the following guidelines (use our free online OligoAnalyzer® tool for this purpose):
    -> The difference between melting temperatures (Tm) of the primers should be less than 5°C.
    -> The GC content should be between 35-80% or equivalent to the product being amplified. -> The Delta G value of any self-dimers, hairpins, and heterodimers  should be weaker (more positive) than -9.0 kcal/mole. Positive numbers indicate that the actual secondary structure shown will not form at all.
    -> Avoid 3' complementarity between the two primers to prevent primer dimers.

    The IDT OligoAnalyzer program can be used to assess these different criteria for a proposed oligo.

  • What are the length limits for Molecular Beacons?
    Molecular Beacons must be between 18-45 bases.  Beacon designs outside of this range require special approval which can be requested from custcare@idtdna.com.
  • In calculating Tms for oligos, whose nearest neighbor values do you use?
    We use Santa Lucia's nearest neighbor values. Read more about nearest neighbor analysis in this document: Calculation of Tm for Oligonucleotide Duplexes.
  • What is CE service?
    CE, short for capillary electrophoresis, is a purity measurement utilizing a denaturing gel filled capillary to assess the proportion of full-length products to truncated products present in the final oligo sample. HPLC or PAGE purified oligos up to 60 bases in length receive a complimentary CE trace to document final purity. Read more about CE in the Tech Vault document entitled Capillary Electrophoresis of Oligonucleotides.
  • Which oligo purification method would minimize contamination with environmental DNA?
    Since all of our oligos are chemically synthesized (versus isolated from an organic source), our oligos should only contain the specific sequence you request (plus a small percentage of n-1mer internal or truncation products) and there should be no external DNA contamination. For oligos longer than 50 bases, we recommend either PAGE or IE-HPLC purification to reduce the amount of truncated and deletion products in the oligo.
  • Where on a nucleotide are Cy dyes attached?
    5′ and 3′ Cy dyes are attached to the hydroxyl group of the ribose via a phosphodiester bond. Internal Cy dyes are attached to the backbone via the phosphodiester bonds of the bases between which the dyes occur.
  • What type of purification do I need to select for my 454 sequencing primer synthesis?
    Roche recommends IDT HPLC-purified 454 Fusion Primers to ensure that you get the best data from your sequencing runs. The high purity level of HPLC-purified primers minimizes the risk of mispriming events due to truncations and other errors that may compromise sequencing data quality or the number of viable reads that can be obtained from a single Pico Titer Plate run.
  • What is the typical turnaround time for RNA oligos?
    Desalted RNA oligos are usually shipped in 2–3 business days and RNase-Free HPLC purified RNA oligos often ship in 4–6 business days. Please inquire for turnaround times for 5 µmole and 10 µmole RNA synthesis.
  • What is the structure of the ZEN internal quencher?
    The structure of the ZEN quencher is proprietary to IDT. Read more about the ZEN internal quencher at http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/03/29/modification-highlight-zen-internal-quencher.