This Quick Guide provides DNA Shearing protocols for the Covaris microTUBE and miniTUBE consumables using a Covaris ME220 Focused-ultrasonicator instrument.
Values are nominal values. The tolerances are as follows:
• Temperature ± 5 °C • Sample volume: - microTUBE-15: from 15 to 20 µL, ± 1 µL - microTUBE-50: 55 μL, ± 2.5 μL - microTUBE-130: 130 μL, ± 5 μL - microTUBE-500: 320 μL, ± 10 μL - miniTUBE: 200 μL, ± 10 μL •   Water Level ± 0.5
Sample Guidelines
• DNA input: microTUBE-130 and microTUBE-50 up to 5 μg purified DNA; microTUBE-15 up to 1 μg; microTUBE-500 minimum 320 ng and up to 5 μg. •   Buffer: TE - Tris-EDTA, pH 8.0. • DNA quality: Genomic DNA (> 10 kb).  For lower quality DNA, Covaris recommends setting up a time dose response experiment for determining appropriate treatment times. • WARNING: DO NOT use the microTUBE or miniTUBE for long term sample storage.  Samples should be transferred after processing.
Instrument Setup
• Refer to the instrument manual for complete setup. • DNA Shearing vessels have specific racks and waveguides associated with them.
Instrument Settings
• Recommended settings are subject to change without notice. • DNA fragment representation will vary with analytical systems.  Please carry out a time course experiment based on settings provided in this document to reach desired fragment size distribution ( Appendix C ). • For the current printable version of this protocol (PN 010349) 1
Vessel
See Appendix B for screenshots of protocols entered into SonoLab 8.0.2 or higher.
8 microTUBE-130 AFA 8 microTUBE-130 AFA
See Appendix B for screenshots of protocols entered into SonoLab 8.0.2 or higher. Vessel 4 microTUBE-15 Vessel
To ensure reproducible DNA shearing, it is required to centrifuge microTUBE-15 before processing.  See Appendix A for instructions.
microTUBE-50 with SonoLab 8.0.1* or lower

Vessel microTUBE-50 AFA Fiber Screw-Cap (PN 520166)
*For SonoLab version 8.0.2 or higher, refer to the prior table ( page 3 ) for PN 520174 and PN 520240. For all versions of SonoLab, use these protocols for PN 520166.  To upgrade SonoLab, visit our website: https://covaris.com/resources/registered-users-login/m-series/ 6
microTUBE-130 with SonoLab 8.0.1* or lower
8 microTUBE-130 AFA 8 microTUBE-130 AFA Fiber Strip V2 Fiber H Slit Strip V2 microTUBE-130 AFA Fiber Screw-Cap 130 μl
*For SonoLab version 8.0.2 or higher, refer to the prior table ( page 4).  To upgrade SonoLab, visit our website: https://covaris.com/ resources/registered-users-login/m-series/ Vessel Sample Volume 7 microTUBE-130 microTUBE AFA Fiber Vessel Sample Volume 130 μl
microTUBE-500 microTUBE-500 AFA Fiber Screw-Cap (PN 520185)
To fragment DNA to sizes larger than 5 kb, Covaris offers the g-TUBE: a single-use device that shears genomic DNA into selected fragments sizes ranging from 6 to 20 kb. The only equipment needed is a compatible bench-top centrifuge. Vessel 9 miniTUBE miniTUBE Red (PN 520066)
Vessel Additional Accessories

Appendix A: microTUBE-15 Centrifugation before DNA Shearing
1. Sample loading and centrifugation: -   microTUBE-15 AFA Beads Screw-Cap: Load and centrifuge microTUBE-15 Screw-Cap as described before placing the tubes in the rack. If some of the sample splashes onto the wall of the microTUBE while removing from centrifuge or placing into rack, repeat centrifuge step.  All liquid should be at the bottom of the microTUBE-15 before starting the AFA treatment. 8 microTUBE-15 AFA Beads Strip V2: The 8 microTUBE-15 AFA Beads Strip V2 will fit into the Covaris Centrifuge 8 microTUBE Strip V2 Adapter (PN 500541) for the Thermo Scientific™ mySPIN™ 12 mini centrifuge.  Place the strip in the adapter and spin for a minimum of 1 minute. 2.  Sample Processing: - Use settings provided on page 5. 3. Sample Recovery: unscrew the cap. the beads aside for full recovery.
Refer to the ME220 User Manual for detailed instructions on method creation: https://covaris.com/wp-content/uploads/pn_010325.pdf
1.  Method Editor:
-   As an example, below is a screenshot of the 150 bp protocol for the microTUBE-50 in the Method Editor with all samples selected.  NOTE: Repeat ("Iterations") is programmed before the Process.
2.  Shearing Protocol:
Screenshot of a complete shearing protocol with all samples selected for treatment. Appendix C: Performing a Time Course to Create a Pulsing Method using SonoLab 8.0.1 or higher Refer to the ME220 User Manual for detailed instructions on method creation: https://covaris.com/wp-content/uploads/pn_010325.pdf
1. Time Course:
- A time course is necessary to optimize shearing protocols based on user needs.  Covaris recommends using the maximum number of samples (N = 4 for screw-cap consumables or N = 8 for strip or snap-cap consumables) for each time point in the time course.  The time course shall be performed by starting with the nearest recommended protocol (containing "Z" iterations) and increasing or decreasing the total number of iterations used to adjust total treatment time.
2.  Time Course Set-up:
a)  Fill Covaris consumable with lambda or genomic DNA in desired buffer at a concentration of 10 ng/μL or higher - Recommended buffer: Tris-EDTA b) Input protocol settings into SonoLab with a value of Z – 1 iterations or Z – 2 iterations to begin: - I.e. Input method with 2 less iterations than stated for methods shearing to below 200 bp - I.e. Input method with 1 less iteration than stated for methods shearing to above 200 bp c) Insert tubes into the ME220 and run protocol d) Once the treatment is complete, remove samples from the instrument e) Transfer a 5 μL aliquot of each sheared sample to a Lo-Bind vessel for sizing analysis f)  Retain used Covaris consumable and immediately replace the 5 μL missing volume with buffer for a full volume after each method is complete -  i.e. microTUBE-50 would initially be filled to 55 μL for shearing.  5 μL would be removed post-shearing for a total volume of 50 μ L. Buffer is added for a total volume of 55 μ L for next time point. g) Once Covaris consumable volume has been restored, adjust method iterations: - Input method with 2 iterations TOTAL for methods shearing to below 200 bp - Input method with 1 iteration TOTAL for methods shearing to above 200 bp h) Repeat steps c through g. i)   If extra time points are necessary, a new consumable (Strip) or consumables (Snap-Caps, etc.) must be used (see flowchart below for details). j) Retain total iterations or duration(s) used to obtain desired fragment sizes for programming into SonoLab and future use. NOTE: Do not alter settings when running a time course.  Only iterations should be altered.
Workflow for targets lower than 200 bp.  The green box indicates the Quick Guide protocol settings.  The column of orange boxes indicate treatment settings that should be programmed into the ME220 during the time course.  The column of blue boxes indicate settings to be programmed when the target base pair size has been achieved.  The orange text indicates user intervention.
NOTE: New Consumable(s) need to be used for additional time points. Workflow for targets lower than 200 bp.  The green box indicates the Quick Guide protocol settings.  The column of orange boxes indicate treatment settings that should be programmed into the ME220 during the time course.  The column of blue boxes indicate settings to be programmed when the target base pair size has been achieved.  The orange text indicates user intervention.
Technical Assistance
• By telephone (+1 781.932.3959) during the hours of 9:00 a.m. to 5:00 p.m., Monday through Friday, United States Eastern Standard Time (EST) or Greenwich Mean Time (GMT) minus 05:00 hours •   By e-mail at ApplicationSupport@covaris.com
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