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Tips for resuspending and diluting your oligonucleotides

You have received your custom oligonucleotides, and now it is time to resuspend and dilute them. Here are a few tips from our scientists that will help facilitate use of the oligos in your experiments.

Mar 31, 2017

Revised/updated Sep 22, 2017

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.

Below are some useful recommendations from our scientists. For guidance on oligonucleotide storage, see the article, Storing oligos: 7 things you should know.

  • 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 (see Figure 1) on the Oligo Entry ordering tool. Here, you can select LabReady (100 µM in IDTE, pH 8.0), or create a custom formulation to your specifications.
  • 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.

art130a-LT-CORE-Resuspension-Fig1

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. (If yield is 9 nmol, 90 µL of buffer is needed to make a 100 µM solution.)

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:

  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.

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

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.

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