This Quick Guide provides DNA Shearing protocols when using microTUBE-130, microTUBE-50, microTUBE-15, microTUBE-500, or miniTUBE and a Covaris E220 Focused-ultrasonicator.
Values mentioned in this Quick Guide are nominal values. The tolerances are as follows: Temperature +/-2°C - - Sample volume o o o microTUBE-15: from 15 to 20 µl, +/- 1 µl microTUBE-50: 55 μl, +/- 2.5 μl microTUBE Plate, Strip, Snap and Crimp Cap: 130 μl, +/- 5 μl microTUBE-500: 500 μl, +/- 10 μl or 320 μl, +/- 10 μl miniTUBE: 200 μl, +/- 10 μl o o Water Level +/- 1 -
Sample 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 holders or racks associated with them. - E220 and E220 evolution may require the Intensifier (PN 500141). Refer to Appendix C for instructions. - E220 and E220 evolution may require Y-dithering. Refer to Appendix A 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 (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  010308.pdf for updates to this document.
available on SonoLab versions 7.3 and up. Please see Appendix A for detailed instructions. Part Number:
To ensure reproducible DNA shearing, it is required to centrifuge samples before processing DNA in a microTUBE-15.  Please see Appendix B for instructions. Please note that microTUBE-15 requires removal of the Intensifier (PN 500141) from the E220 focused- ultrasonicator. Please see Appendix C for instructions.
Please note that miniTUBE requires removal of the Intensifier (PN 500141) from the E220 focused- ultrasonicator. 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

A Y-dithering step is required for DNA shearing with the 96 microTUBE-50 Plate - - - 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):  0.5 b.   X-Y Dither Speed (mm/sec):  10.0 Both X Dither (mm) and X-Y Dwell (sec) should be set to O C.

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 in page 4.
3. Sample recovery
Repeat the centrifuge step before recovering sample from microTUBE-15. Preparation Station and unscrew the cap
The 500141 Intensifier is a small inverted stainless steel cone centered over the E Series transducer by four stainless wires.  The wires are held by in a black plastic ring pressed into the transducer well. If an AFA protocol requires “no intensifier”, please remove the Intensifier, using the following steps: Empty the water bath.  Start the E System and start the SonoLab software. 1. Wait for the homing sequence to complete (the transducer will be lowered with the rack holder at it home position, 2. allowing easy access to the Intensifier). Grasp opposite sides of plastic ring and gently pull the entire assembly out of the transducer well.  Do not pull on the steel 3. cone or the wires.  The ring is a friction fit in the well – no hardware is used to hold it in place. The 500141 Intensifier (left) shown installed in the E System transducer well and (right) removed. Note the “UP” marking at the center of the Intensifier. If a protocol requires the Intensifier to be present, simply reverse this process: Align the black plastic ring with the perimeter of the transducer well.  Note that the flat side of the center cone (marked UP) 1. should be facing up (away from the transducer). 2. Gently press each section of the ring into the well until the ring is seated uniformly in contact with the transducer, with approximately 2 mm of the ring evenly exposed above the transducer assembly.  Do not press on the cone or wires.  The rotation of the ring relative to the transducer assembly is not important. Refill the tank.  Degas and chill the water before proceeding. 3.
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 .