This Quick Guide provides DNA Shearing protocols when using microTUBE, microTUBE-50, microTUBE-15, microTUBE-500, or miniTUBE and a Covaris LE220 Focused-ultrasonicator.
Values mentioned in this Quick Guide are nominal values. The tolerances are as follows: Temperature +/-2°C - Sample volume - microTUBE-15: from 15 to 20 μl, +/- 1 μl o microTUBE-50: 55 μl, +/- 2.5 μl o microTUBE Plate, Strip, Snap and Crimp Cap: 130 μl, +/- 5 μl o microTUBE-500: 320 μl, +/- 10 μl o miniTUBE: 200 μl, +/- 10 μl o Water Level +/- 1 -
Sample preparation guidelines
- DNA input: up to 5 μg purified DNA (1 μg for the microTUBE-15; minimum 320 ng for the microTUBE-500) - Buffer: 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. DO NOT use the microTUBE or miniTUBE for storage. Samples should be transferred after processing. -
Instrument setup
Refer to the instrument manual for complete setup. - - microTUBE and miniTUBE have specific racks associated with them. LE220 protocol may require X and/or Y-dithering. Refer to Appendices A and B for instructions. -
Instrument settings
- Recommended settings are subject to change without notice. Mean DNA fragment size distributions are based on electropherograms generated from the Agilent Bioanalyzer - with the DNA 12000 Kit (cat# 5067-1509), with the exception of the 320 μl microTUBE-500 protocol (Agilent High Sensitivity DNA Kit, cat# 5067-4626). 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. See http://www.covaris.com/wp-content/uploads/pn_010156.pdf for updates to this document.
The X-dithering and Y-dithering functions are both required for shearing with the 8 microTUBE-50 AFA Fiber Strip V2 and the 96 microTUBE-50 AFA Fiber Plate. These functions are only available on SonoLab versions 7.3 and up. Please see Appendix A for detailed instructions.
The Y-dithering function is required for shearing with 15 μl samples. This function is only available on SonoLab versions 7.3 and up. Please see Appendix B for detailed instructions. To ensure reproducible DNA shearing, it is required to centrifuge samples before processing DNA in a microTUBE-15. Please see Appendix C for instructions.
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 kb to 20 kb. The only equipment needed is a compatible bench-top centrifuge.

Additional Accessories

Technical Assistance By telephone (+1 781 932 3959) during the hours of 9:00am to 5:00pm, Monday through Friday, United States Eastern . Standard Time (EST) or Greenwich Mean Time (GMT) minus 05:00 hours . By e-mail at techsupport@covaris.com
X and Y-dithering are required for DNA shearing with the 8 microTUBE-50 AFA Fiber Strip V2 and 96 microTUBE-50 AFA Fiber Plate - - - - This feature is only available on SonoLab versions 7.3 and up. There are dithering limitations on instruments with serial numbers below 2000. To obtain a copy of the SonoLab 7.3 and the Plate Definition installers, please employ the Registered Users Login on the Covaris website, www.covaris.com For any assistance in this process, please contact your local representative, or Covaris Global Technical Services at TechSupport@covaris.com. Use the following steps to include X-dithering and Y-dithering in sample treatment: 1.   Go into the Method Editor 2.   Select ‘Add Step’ and enter the treatment settings for the desired fragment size a.   Note: The following steps must be done for each individual treatment 3.   Select the Motion tab 4.   Enter the following values into the ‘X-Y Dithering’ box a.   X Dither (mm):  0.5 b.   Y Dither (mm): 0.5 c.   X-Y Dither Speed (mm/sec):  10.0 d.   X-Y Dwell (sec) should be set to O Appendix B – Using Y-dithering with SonoLab 7.3 and up Part Number: 010156 Rev R
Y-dithering is required for DNA shearing with the microTUBE-15
- - - This feature is only available on SonoLab versions 7.3 and up. To obtain a copy of the SonoLab 7.3 and the Plate Definition installers, please employ the Registered Users Login on the Covaris website, www.covaris.com For any assistance in this process, please contact your local representative, or Covaris Global Technical Services at TechSupport@covaris.com. Use the following steps to include Y-dithering in sample treatment: 1.   Go into the Method Editor 2.   Select ‘Add Step’ and enter the treatment settings for the desired fragment size a.   Note: The following steps must be done for each individual treatment 3. Select the Motion tab 4. Enter the following values into the ‘X-Y Dithering’ box a.   Y Dither (mm):  5.0 b.   X-Y Dither Speed (mm/sec):  20.0 c.    Both X Dither (mm) and X-Y Dwell (sec) should be set to O

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 4.
3. Sample recovery
Repeat the centrifuge step before recovering sample from microTUBE-15. the cap narrow bore 20 µ L pipet tip. It may be necessary to push the beads aside for full recovery