Sanger Tree of Life HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio

Label the required number of Diagenode Hydro Tube for each DNA sample that will be sheared; ensure to label the tubes both on the lid and on the side.

Prior to transferring the DNA sample from its original tube, first mix the DNA sample by pipetting carefully with wide-bore pipette tip.

Transfer the desired amount of each DNA sample to their corresponding labelled Diagenode Hydro Tube shearing tube. The volume of DNA to be sheared must be 100–130 µL and at a concentration of 3 ng/µL–39 ng/µL, ideally 30 ng/µL.

Ensure that all the tubes have an equal volume by using EB buffer or nuclease free water to normalise the volumes.

Replace the Hydro Tube lids and briefly centrifuge the tubes using a mini-centrifuge.

Before attaching the Hydropore-Syringe to the top of the tube, check that each syringe is screwed together and ensure that all joints/screw tops are tight and that the plunger is pushed all the way down.

Remove the Hydro Tube lids and fit a Hydropore-Syringe to the top of the tube by pushing firmly (note: it does not screw on). Retain the lid of the Hydro Tube.

Switch on the Diagenode Megaruptor®3 and initialise using the on-screen instructions: follow the prompt to remove the loading cassette, lifting up and away from the base unit, wait approximately 3 seconds for the arms to set the machine to ‘zero’ position, and then replace the cassette on the base unit.

Once initialised, use the touch screen to set up the run; select “Protocols” and then “Go & Shear''. Set the parameters as follows:

1. Concentration: select the highest concentration in the sample set. Note: Concentrations in the same run should not vary more than ± 25 ng/µL, and samples with concentrations below 50 ng/µL should not be combined on the same run as those above ng/µL. The Megaruptor should not be used to shear samples with concentrations higher than 150 ng/µL – these samples should be diluted.
2. Volume: as measured
3. Speed: 31

Load the Hydropore-Syringes into the cassette when prompted by touch screen. The Hydropore-Syringes must be balanced, i.e. loaded symmetrically across the 8 positions, with an empty balance Hydropore-Syringe used where there is an uneven number of samples. When loading onto the instrument, orientate the Hydropore-Syringe so that the clear side faces out to allow the DNA solution to be observed moving up and down.

After starting the program, visually confirm that all syringes are drawing up liquid. If a sample is not moving through the hydropore once the “shearing” step is reached there may be an issue. See the ‘Troubleshooting’ section in the Guidelines section for details on how to deal with any difficult samples.

The run takes approximately 45 mins. Be aware the displayed countdown on the machine is not accurate.

Once the program is complete, remove each Hydropore-Syringe from the instrument.

Remove the Hydropore-Syringe from the Hydro Tube, pressing down on the plunger during removal to ensure the collection of any residual liquid/sample within the hydropore. Refer to the ‘Maximising Sample Retrieval’ for tips on how to minimise sample loss within the Hydropore-Syringe.

Discard the Hydropore-Syringes as biological waste and replace the retained lids onto the Hydro Tubes.

Store the sheared DNA at 4 °C until further processing.