TechVault
  • Why do my OD measurements not match the values reported by IDT?
    OD values are easily affected by a number of small variables. IDT takes two separate OD measurements to ensure accuracy of the OD calculation. These values are then averaged and a final OD value is reported. Depending upon the machine used, the sample prep conditions, and the algorithm applied to determine the OD values, there will be some variation from method to method. OD values reported by IDT should be within 20% of the actual OD value for the product depending upon the preceding variables.
  • Where can oligo invoices, quality control documents, and specification sheets be found online?
    For orders placed via the web, all of these documents are uploaded to your Order History. Quality control documents can be downloaded in ZIP format or individually by expanding the particular order with the plus sign on the left hand side of the screen. A Certificate of Analysis containing specifications for the entire order is avail- able in the Order History. Individual specification sheets can be downloaded the same way as individual quality control documents. Finally, invoices are also available in PDF format through Order History. Please note: if you do not see an older order in the history, you may need to expand the date range using the Search link in the upper right hand corner
  • What is the best way to purify PCR products?
    For most applications, it is best to purify PCR products by gel electrophoresis. This simple method not only purifies the final PCR product, but can be a valuable troubleshooting tool as well since nonspecific PCR products, primer-dimer products, negative amplification, and other elements can easily be identified by gel analysis.
  • For which applications is it better to order Ultramer™ sequences vs. short oligonucleotides?

    The Ultramer product utilizes an enriched synthesis chemistry that has a higher coupling efficiency, and as a result, you receive a higher percentage of full-length product in your sample. These higher purity sequences are suitable for demanding applications such as cloning, ddRNAi (DNA directed RNAi), and gene construction and can save researchers a great deal of time and trouble in these applications through direct synthesis of the entire target fragment. Additionally, Ultramer oligonucleotides are a good choice for template synthesis due to the available lengths of up to 200 bases.

  • How do I enter RNA sequences online?
    Oligonucleotides containing at least one RNA base must be ordered from our Custom RNA Oligos ordering page.  RNA bases are entered with a lower-case “r” preceding the desired base (e.g., rA, rC, rG, rU).  For your convenience, the “Convert to RNA” button under the sequence entry box will add a lower-case “r” in front of all bases and change all T bases to U bases. Please note that you can use this same method to order RNA/DNA chimeras. To denote the DNA bases, simply use the capital letter of the desired base type (e.g., A, C, G, T). There is no extra charge for DNA bases in an RNA sequence beyond the standard per base charge for DNA on the scale requested.
  • When is it important to get HPLC purification?
    HPLC purification helps remove truncated synthesis products and enrich purity. Purification is recommended for oligos greater than 40 bases in length and for many modified oligos. In addition, many modifications require HPLC purification due to the nature of their synthesis chemistries. HPLC purification typically results in approximately 85% purity. Purity guarantees are issued on sequences up to 60 bases as long as they are not heavily modified or do not contain significant secondary structures. In general, HPLC tends to produce oligonucleotides that are slightly less pure than PAGE, but typically, with a higher guaranteed yield. We offer two different types of HPLC purification: Reverse Phase HPLC (RP-HPLC) and Ion Exchange HPLC (IE-HPLC). IE-HPLC is useful for oligos that will be transfected into living cells, while RP-HPLC is needed to purify oligos with fluorescent dyes and some other hydrophobic modifications. For those oligos that require the use of RP-HPLC purification and are to be used in living cells, we recommend following RP-HPLC with a Na+ salt exchange to remove any potentially toxic buffers such as triethylammonium acetate (TEAA). Please note that for very long sequences, over 80 bases, we recommend PAGE purification. 
  • What is your protocol for annealing oligos?

    It is sometimes necessary to make double-stranded DNA from single-stranded oligos. While the annealing procedure is simple, attention to a few details can greatly reduce the presence of undesired single-stranded material.

    Method:

    Dissolve each oligo in “Duplex Buffer” (100 mM Potassium Acetate, 30 mM HEPES, pH 7.5) at high concentration (1−10 OD260 units/100 μL). The presence of some salt is necessary for the oligos to hybridize.

    1. Mix the two sequences together in equal molar amounts. If different amounts are used, there will always be single-stranded sequence left over.
    2. Heat to 94°C and cool gradually. For many oligos this can be as simple as transferring from 94°C to the bench-top (room temperature). For sequences with significant hairpin potential, a more gradual cooling/annealing step is beneficial; this is easily done by placing the oligos in a water bath or heat block and unplugging the machine.
    3. The resulting product will be in stable, double-stranded form and can be stored at 4°C or frozen.

    Additional tips:

    If the product will be used in a ligation reaction, the addition of 5’-phosphate may be needed. This can be done at the time of oligo synthesis (chemical phosphorylation) or at any time thereafter using PNK (enzymatic phosphorylation). If the oligos are relatively long or will be used in cloning, starting with PAGE-purified oligos is recommended.

  • How do I order modified oligos online?
    Modified oligos can be ordered from our Custom DNA Synthesis or Custom RNA Synthesis page. Modifications can be added to the sequence from the modification tabs below the sequence entry box. Not all modifications are available for all synthesis scales. If you do not see the modification you are looking for, adjust the oligonucleotide synthesis scale.
  • How many reactions will I be able to do with 25 nmole PCR primers?
    Most PCR reactions use 0.1−0.5 μM primer. Assuming a maximum concentration of 0.5 μM and a reaction volume of 20 μL, each reaction will require 10 pmoles of oligonucleotide primer. For a typical 25mer oligonucleotide, 1 OD is equivalent to approximately 4 nmoles, or 4000 pmoles. Guaranteed yield for most constructs on the 25 nmole scale is 3 ODs. Therefore, with even the minimum yield from a 25 nmole synthesis, you should be able to perform 1200 PCR reactions. In summary: 3 ODs of a 25mer ≈ 12 nmoles = 12,000 pmoles = 1200 reactions.
  • How do I order an oligonucleotide with a modification?
    When ordering, select the Custom DNA Oligos option, and then the type of oligo product you desire. At the bottom of that ordering page you will see tabs that read 5’ Mods, Internal Mods, and 3’ Mods. Most modifications are not available on the 25 nmole scale, so if you do not see the modification you are looking for, increase your synthesis scale to at least 100 nmol. This will increase the menu of modification options available. When you find the modification you want, click on the “Add” button on the left, which will add the appropriate code to your sequence.
  • What are OligoCard payment cards?
    OligoCard® payment cards are prepaid cards that can be used to purchase IDT products. They require only a single purchase order or credit card transaction from your purchasing group. This one-time payment can then be used as needed for IDT purchases. OligoCard payment cards can be ordered online. Once ordered, you will receive a secure PIN that you can use to unlock your credit online. Once activated, you can use these cards in place of a PO or credit card as payment. OligoCard histories and balances can be managed through the web. Also see the OligoCards Catalog Page. 
  • What if I need a gBlocks Gene Fragment library that is more complex than what you offer online?

    We realize that this first offering for gBlocks Gene Fragments libraries is somewhat limited. We are working hard at offering more complex libraries in the future. Help us prioritize by sharing what you need from us in as much detail as possible (i.e., show us sequences including mixed bases, annotations, drawings, etc.) and send to libraries@idtdna.com.
  • Does IDT provide a negative control sequence for miRNA Inhibitor (anti miRNA oligonucldoetide, or AMO) experiments?
    Yes. A good negative control should be inert and not modulate any genes in the system under study. This is difficult to achieve; however, we propose 2 negative control sequences that we have used throughout our product development and validation process, and which we have found to work very well in vitro and in vivo. You can read more about as well as order positive and negative controls for miRNA Inhibitor experiments under the "Support" tab of our miRNA Inhibitor product page.
  • Why am I limited to 18 N or K mixed bases in my gBlocks Gene Fragment?

    Incorporating 6 NNK codons corresponds to about 1 billion possible combinations, and 18 N mixed bases will create a pool with 68.7 billion different gene fragments. As the number of variable bases increases, the number of molecules representing a particular sequence decreases. We decided to limit variable regions to 18 mixed bases to give customers the best overall pool of library constructs. Since most functional screens cover 1,000–100,000 recombinant colonies, allowing 18 mixed bases should be adequate for producing meaningful results.

  • What secondary structure considerations need to be included when designing primers for PCR?

    When designing primers for PCR, two types of secondary structures should be analyzed: dimers and hairpins. For dimers, both self- and hetero-dimers should be reviewed. In general, the ΔG value for dimer analysis should be between 0 to −9 kcal/mole for optimal design. Values more negative than this may adversely affect PCR reactions. For hairpins, the Tm of the hairpin should be lower than the annealing temperature for the reaction. The Tm for the strongest hairpin should be at least 50°C below the annealing temperature. These secondary structures and their corresponding stabilities can be calculated online with the IDT SciTools® OligoAnalyzer program

  • How can I determine the guaranteed yield for a specific construct?

    Guaranteed yield is the minimum amount of oligonucleotide IDT will deliver and is dependent upon sequence composition, modifications, and purification method. The best way to determine the guaranteed yield for a specific oligonucleotide is to add that construct to the shopping cart on the website. Once you have added an oligonucleotide to your shopping cart, you will see its guaranteed yield in ODs, nmoles, and μg. If the guaranteed yield is not sufficient for your application, you can adjust the starting scale using the Edit link in the shopping cart.

  • Can I get custom ratios of random (mixed, degenerate) bases incorporated?

    IDT offers two types of randomization, machine mix and hand mix. Machine mix bases are charged at the standard base price for the scale ordered. Hand mixing is done to provide custom ratios of the bases, and incurs an additional charge. When entering your sequence, the “Mixed Base” tab at the bottom of the page lists the IUB symbols and is where custom mix ratios need to be entered.

  • How do I reorder an oligo?
    For IDT orders placed through the web, reorder items via Order History.  Access Order History using the Order dropdown menu on the IDT website.  Search for specific items and add them to the shopping cart for reorder.
  • What are the N and K mixed bases that can be incorporated into the variable regions of gBlocks Gene Fragments?
    Amino acids are represented by 1–4 codons that are defined by the genetic code. NNK codons are commonly used in screens for codon substitutions to reduce the presence of some codon-rich amino acids, thereby reducing their overrepresentation in a particular library. Additionally, using NNK codons removes 2 out of 3 possible stop codons, which also limits the number of sequences in a library that produce unwanted truncated gene products.
  • Can I order multiple variable regions in my gBlocks Gene Fragment?
    Currently, only consecutive bases are allowed. Specifically, up to 18 consecutive N or K bases can be ordered, with a minimum of 125 bp of fixed, flanking sequence on either side of the variable bases. The maximum length for the entire sequence is currently 500 bp, including the variable bases.