DNA shearing with the qSonica Sonicator

Before using the qSonica instrument for the first time, consult with Athena or Grace for proper training.

Sonication time varies by DNA size, genome size, tube volume, and sample buffer. Before you begin, please consult with Athena for the best trial conditions to select based on other recent results. Additional user validated protocols can be found here.

Protocol

  1. Thaw samples, if necessary, and place on ice.
  2. Ensure all tubing is properly connected.
  3. Add ~1.5 L cold DI water to the qSonica bath compartment (if empty).
  4. Turn on the water cooler and allow the system to cool to 4 °C. If the bath is filled with room temperature water, it will take 15-20 minutes to cool.
  5. Adjust the water adjustment dial (“-” direction) until the rear reservoir bladder is appx. half full. The water level inside the bath should be ~2 cm above the titanium pedestal. If not, add water.
  6. Turn on the power supply (“|” icon on the top right). Ensure that the sonication bath is empty and that the cabinet door is latched closed.
  7. Run the “degas” program (appx. 10 minutes). While degassing, complete steps 8 and 9.
  8. Transfer samples to Brandtech 0.2 mL tubes. Sonication tube volume should always be identical within each batch; add molecular-grade water as required.

  9. Load samples into the blue 18-place tube holder. If you have fewer than 18 samples, fill empty spaces with blank tubes containing water (blanks are kept in the drawer). Cover with the white donut and screw on the top section. Leave the assembly on ice or at 4°C until ready for use.

    You can use the orientation of the tube hinges to make it easy to distinguish samples from blanks when tube holder is assembled and in the machine. In the example below, blanks are loaded with hinges facing inward (far left), whereas samples are loaded with hinges facing outward (center left) before adding the “donut” (center right) and top section (far right). qSonica rosette assembly

  10. After degassing, use the controls on the power supply to set the sonication conditions. Example: Timer = 2:30 (total sonication on time); Pulse = 15s ON / 15s OFF; Amplitude = 40%.

    There is a vortex and spin step halfway through sonication. Set the timer to half the actual sonication time, vortex, and repeat the cycle once.

    In all cases qSonica and users report the “Total Sonication ON Time”. A protocol using a pulse such as 15s on/15s off will take twice as long to complete.

  11. Attach the sample rack to the lid of the sonicator bath. Make sure the lid is plugged in.

  12. Visually inspect the bath water level. Adjust the water adjustment dial if it is too high or too low.

    The water level in the bath should match that of the water level in the sample tubes as closely as possible. If they are not a perfect match, it is better if the water level of the bath is slightly below the water level of the sample tubes. Otherwise, excessive splashing may occur.

  13. Close the cabinet door and use the red start/stop button to start the sonication run. Check that you see only minor splashing during the first ON cycle. (A slight misting is fine, but large droplets are not.) You can adjust the water level while sonication is proceeding if small adjustments need to be made.
  14. Halfway through the sonication protocol, stop sonication, remove the tube rotator from the instrument, and vortex the tubes (easiest to do while they are still in the rotator.) Then remove the tubes from the blue 18-place tube holder and spin down any splashing in your tubes. This will result in more even sonication and very little residual HMW DNA.
  15. Resume sonication. You may have to make small adjustments to the water level again.
  16. After sonication is complete, open the cabinet and remove the sample rack. Spin down tubes and either proceed directly to bead cleaning or store frozen until ready to proceed.
  17. When optimizing, after sonication, take an aliquot (~5 μL) sample to run on an agarose gel to assess the sizing pattern. This is not necessary once the sonication parameters have been worked out for your project. If you assess some samples in a set but not others, spike in the same amount of liquid you remove (EB or water) to keep the volumes equal.
  18. Tips for gel electrophoresis:
    • Pour a 1.5-2.0% gel to better visualize smaller fragments
    • Use fresh running buffer rather than recycled (which may contribute background which may be difficult to distinguish from your sonicated material
    • Flank your sonicated samples on both sides of the row with a ladder (instead of just using one). This will make it easier to assess the samples in the middle of the row.

    With most library types, an ideal sonication result is to have the brightest part of the smear overlap with the 300-500 bp range and to have only trace amounts of material greater than 1,000 bp (see example below). qSonica gel

  19. Once general conditions are worked out for a project, continue with the next set of tubes to sonicate. When you are done for the day, turn off the chiller pump and use the “0” button on the power supply to turn it off. Consult with the lab manager about when and how to empty the water from the bath.
  20. Optional: assess all project samples on a gel. (note: only those starting with 500 ng+ will be easily visible.) Add extra sonication time to any that are outliers.
  21. Completed samples may be stored at -20°C while sonication for a project is on-going.