Oligo Handling, Analysis, and Applications
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Tips for resuspending and diluting your oligonucleotides

Oligo resuspension

If you receive your oligos dry, you will need to resuspend them in aqueous solution before use. Most oligos are relatively easy to resuspend; however, those modified with fluorophores or hydrophobic molecules may require more time to become completely solubilized.

Here are some useful recommendations from our scientists:

  • During the dry-down process, oligos form a white flakey pellet at the bottom of the tube. Since the pellet can become dislodged during shipping, it is crucial to briefly centrifuge every oligo before opening the tube. Failure to do so could result in yield loss, because oligo pellets that are not at the bottom of the tube could fly out of the tube when the cap is opened.
  • If resuspension is difficult, try heating the oligo at 55°C for 1–5 minutes, then vortex thoroughly. If any precipitates remain, they are likely either trityl groups (flakey appearance) or the controlled-pore glass (CPG; a pellet at bottom of tube) on which the oligo was synthesized. Neither should affect oligo performance, and both can be removed—just run the resuspended oligo through a Sephadex® G-50 column (GE Healthcare) or transfer the supernatant (which contains the oligo) to a new tube.
  • IDT oligonucleotides (both DNA and RNA) are typically shipped dry. However, you can request to have your DNA oligos resuspended prior to shipment. To obtain resuspended oligos, use the Normalization dropdown menu on the Oligo Entry ordering tool (see the heavy red box on the right, in Figure 1). Here, you can select LabReady (100 µM in IDTE, pH 8.0), or create a custom formulation to your specifications.

  • Oligo ordering screen

    Figure 1. Request your oligos be sent resuspended from the Custom DNA Oligo ordering screen.

  • Do not expose your oligos to highly acidic or basic conditions at any time during resuspension. We recommend resuspending oligos in a TE buffer solution, such as IDTE, to maintain a constant pH that supports oligo stability (IDTE is available from IDT at pH 7.5 and pH 8.0). Alternatively, resuspend oligos in nuclease-free, sterile water, pH 7.0 (HPLC-grade is preferable; available from IDT).

  • Note: For long-term storage, avoid resuspending oligos in DEPC-treated water or water from deionizing systems. These are often acidic (pH as low as 5) and may cause DNA degradation over time.

Storage concentration

Be sure to store your oligos at concentrations >1 µM, up to 5 mM. If stored at concentrations ≤1 μM, oligo loss can occur over time due to adsorption into the tube plastic. 5–10 mM is generally the highest concentration at which an oligo can be solubilized.

Aliquoting and diluting oligos

You can reduce the risk of contamination by aliquoting your resuspended oligos. For best practice, prepare a 100 µM “storage” solution and then divide it into a series of tubes for individual use. As alternative concentrations are needed for your experiments, dilute storage solution aliquots to create one or more “working” solutions.

To make a 100 µM storage solution:

  1. Find the oligo yield information (in nmol) on the tube label or specification sheet.
  2. Multiply this number by 10.
  3. The resulting product is the amount of buffer needed, in µL, to prepare a 100 µM solution.

    For example: If yield is 9 nmol, 90 µL of buffer is needed to make a 100 µM solution.

    The unit conversion works out as follows:

    9 nmol/90 µL= 0.1 nmol/µL = 100 pmol/µL = 100 µM

Note: At 100 µM concentration, 1 µL solution contains 100 pmol of oligo. PCRs typically require 10–50 pmol of each primer per reaction.

To make storage solutions at other concentrations:

Alternative storage and working solution concentrations can be made easily using our Resuspension Calculator.

  1. Find the oligo yield information on the tube label or specification sheet. This will be listed in 3 forms—optical density (OD), nanomoles (nmol), and mass (mg).
  2. Enter one of these yield measurements in the Quantity box of the Resuspension Calculator and select the appropriate units.
  3. In the Final Concentration box, enter your desired stock concentration and again select the appropriate units.
  4. Click Calculate. The tool will determine the volume of buffer/water needed to create the desired stock.

Note: Based on the units selected, you may be required to enter the molar extinction coefficient and/or the molecular weight of your oligo. This information can be found on your oligo specification sheet.

To make a working stock from storage stock:

Working stocks can be made easily from storage stocks using our Dilution Calculator.

  1. Enter the starting concentration and volume, then select the appropriate units.
  2. Enter the final (desired) concentration and volume, and select the appropriate units.
  3. Click Calculate. The tool will determine the volume of buffer/water needed to dilute your storage stock to the desired working stock concentration.

Note: Based on the units selected, you may be required to enter the molecular weight of your oligo. This information can be found on your oligo specification sheet.

For further assistance in resuspending and diluting your oligos, please contact our application support specialists at applicationsupport@idtdna.com.

Product focus—Oligonucleotides and primers, dsDNA fragments

Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides, and they will be shipped to you the next business day (larger scales are shipped within 5 business days, pending final quality control). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Learn more or order now.

Custom dsDNA fragments—gBlocks® Gene Fragments

Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

Learn more about gBlocks Gene Fragments at www.idtdna.com/gBlocks.

Additional reading

Which type of oligo purification should I choose?—Do your oligos need purification for your intended application? Use these recommendations based on oligo length, application, yield required, and presence of modifications to determine which oligonucleotide purification method to select.

Oligonucleotide modifications: Choosing the right mod for your needs—Learn about our broad family of oligonucleotide modifications, and get suggestions for selecting modifications that can help you in your research.

RxnReady Oligos—get your oligos premixed!—Have 2–6 standard desalted DNA oligonucleotides premixed in a single tube according to your specifications. This can be useful when performing multiplex PCR, or when generating sets of insertions or deletions through site-directed mutagenesis.

Author: Nolan Speicher is a scientific writer at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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