M 220 Focusedultrasonicator User Training Adaptive Focused Acoustics

M 220 Focused-ultrasonicator User Training

Adaptive Focused Acoustics® (AFA®) John Doe September 00, 2017 www. covaris. com Proprietary 2

The Patented Covaris AFA® Attributes • AFA is the technology behind Covaris Focusedultrasonicators • Solid state 0. 5 MHz ultrasonic transducer • 3 mm wavelength ideal for processing biological molecules • Geometrically focused acoustic energy • Non-contact delivery of Controlled focused acoustic energy • Acoustically optimized sample vessel design • Consistent sample temperature Proprietary 3

How does ultrasonication work? Cavitation • • • Acoustic waves passing through a solution cause localized pressure fluctuations The fluctuations cause dissolved gasses to form microscopic bubbles The bubbles grow, oscillate, and collapse These processes generate shear forces Covaris AFA technology enables precise control of the generated shear forces • Cumulative effect of hundred of thousands of cavitation bubbles Stroboscopic image of single collapsing 2 mm bubble @ 60 Hz Proprietary 4

How does an acoustic treatment work? AFA treatments consist of very high-frequency acoustic signals occurring in a series of “bursts” with each burst followed by a zero power state, as illustrated on the next slide. AFA Treatments are defined by the following parameters: • Cycles per Burst - the number of acoustic oscillations contained in each burst. The illustration on the next slide shows five cycles in each burst. • Duty Factor - the percentage of active burst time in the acoustic treatment. The illustration shows a Duty Factor of 20%. • Peak Incident Power - the instantaneous power, in Watts, driving the transducer during each burst. • Average Incident Power - Peak Incident Power multiplied by the Duty Factor. Proprietary 5

How does an acoustic treatment work? Covaris electronics drive the transducer with a train of electrical “bursts” (t 1) with amplitudes defined by Peak Incident Power (A). Each burst has a duration defined by “Cycles per Burst” and the ON time is defined by Duty Factor (t 1/t 2). Proprietary 6

How does an acoustic treatment work? Nominal average power is the Peak Incident Power multiplied by the Duty Factor. The shape and type of sample vessel, sample volume, water temperature and water quality influence actual power measured by the system and reported by Sono. Lab. Proprietary 7

M 220 Focused-ultrasonicator John Doe September 00, 2017 www. covaris. com Proprietary 8

M 220 - Overview Proprietary 9

System Attributes • • • Runs on Sono. Lab™ software Compact design (computer mount on top of instrument) Low water volume (approximately 15 ml) Degassing not required Integrated temperature control – no external chiller required. All run parameters programmable via Sono. Lab software Proprietary 10

M 220 - Acoustics Sample Holder (Insert) Water Bath Housing Sensor Module Water Level Sensor Transducer 500 k. Hz Proprietary 11

M 220 Holder XTU and Inserts A single XTU holder will accommodate all 4 micro. TUBE inserts. M 220 Holder XTU Insert micro. TUBE 50 μl 500488 M 220 Holder XTU Insert micro. TUBE 130 μ 500489 M 220 Holder XTU Insert micro. TUBE 15 μl 500420 M 220 Holder XTU Insert micro. TUBE 500 μl 500471 M 220 Holder XTU 500414 Proprietary 12

M 220 Holder XTU, Insert, and Consumables M 220 Holder XTU Insert micro. TUBE 15 µl 500420 micro. TUBE-15 Beads Screw-cap 520145 milli. TUBE 1 ml 520128 M 220 Holder XTU Insert micro. TUBE 50 µl 500488 micro. TUBE-50 AFA Filter Screw-cap 520166 milli. TUBE 1 ml AFA Fiber 520130 M 220 Holder XTU Insert micro. TUBE 130 µl 500489 M 220 Holder XTU Insert micro. TUBE 500 µl 500471 M 220 Holder XTU 500414 M 220 Holder XTU Insert micro. TUBE 15 µl 500422 micro. TUBE-130 AFA Filter Screw-cap 520216 micro. TUBE AFA Fiber Crimp Cap 520045 micro. TUBE-500 AFA Fiber Screw-cap 520185 Proprietary 13

M 220 - Features Simple controls and start requirements Easy fill with AFA-grade water Proprietary 14

M 220 – Water Temperature Control Proprietary 15

M 220 – Preloaded Methods Proprietary 16

micro. TUBEs for DNA Shearing Part Number Product Name Sample Volume Cap Fragment Size Beads/Fiber 520145 (25) 520153 (250) microtube-15 AFA Beads Screw-Cap 15 to 20 µl, +/- 1 µl Screw-Cap 100 bp - 1. 5 kb Beads 520166 (25) 520167 (250) micro. TUBE-50 AFA Fiber Screw-Cap 55 µl, +/- 2. 5 µl Screw-Cap 100 bp - 1. 5 kb Fiber 520216 (25) micro. TUBE-130 AFA Fiber Screw-Cap 130 µl, +/- 5 µl Screw-Cap 100 bp - 1. 5 kb Fiber 520185 (25) micro. TUBE-500 AFA Fiber Screw-Cap 320 µl, +/- 10 µl 500 µl, +/- 10 µl Screw-Cap 100 bp - 1. 5 kb Fiber Proprietary 17

mini. TUBEs The Covaris mini. TUBE is optimized to reproducibly shear genomic DNA into fragments ranging from 2 to 5 kb in size. Fragment distributions are tight and precisely centered on the targeted size with three available classes of mini. TUBEs. • Dynamic DNA mass range of 1 µg to 10 µg • Mate Pair libraries • Raindance library prep Clear mini. TUBE 520064 Fragments centered on 2 kb Blue mini. TUBE 520064 520065 Fragments centered on 3 kb Red mini. TUBE 520066 Fragments centered on 4 kb Use M 220 holder P/N 500302 for mini. TUBEs Proprietary 18

tru. Ch. IP Consumable – Kits and Tubes Part Number Product Name Sample Volume 520083 (25) tru. Ch. IP Chromatin Shearing Kit Tissue SDS • Includes low tissue mass optimized protocol for processing up to 20 mg of tissue in 130 µl shearing volume • Includes high tissue mass optimized protocol for processing up to 120 mg of tissue in 1 ml shearing volume 520154 520155 (250) tru. Ch. IP Chromatin Shearing Reagent Kit with Formaldehyde • Includes low-cell protocol optimized for processing 0. 5 -3 million cells in 130 µl shearing volume • Includes high-cell protocol optimized for processing 5 -30 million cells in 1 ml shearing volume 520156 tru. Ch. IP Ultra-low Chromatin with Formaldehyde • Optimized for processing ≤ 100 K cells Part Number Product Name Sample Volume Cap Fragment Size Beads/Fiber micro. TUBE-130 AFA Fiber Screw-Cap 130 µl, +/- 5 µl Screw-Cap 100 bp - 1. 5 kb fiber 520128 (100) 520129 (1000) milli. TUBE 1 ml Screw-Cap 200 -700 None 520130 (100) 520131 (1000) milli. TUBE 1 ml AFA Fiber 1 ml Screw-Cap n/a Fiber 520216 (25) Proprietary 19

Validated Protocols for micro. TUBE 200 bp: PIP 50 W, DF 20%, cpb 200, time 160 sec, temp 20ºC Average 202 bp Std Dev 4. 3 bp CV 2. 1% Proprietary 20

Validated Protocols for micro. TUBE 200 bp: PIP 50 W, DF 20%, cpb 200, time 160 sec, temp 20ºC Average 296 bp Std Dev 6. 4 bp CV 2. 2% Proprietary 21

Validated Protocols for micro. TUBE 500 bp: PIP 50 W, DF 10%, cpb 200, time 60 sec, temp 20ºC Average 496 bp Std Dev 21. 7 bp CV 4. 4% Proprietary 22

Validated Protocols for micro. TUBE 1000 bp: PIP 50 W, DF 5%, cpb 200, time 60 sec, temp 20º C Average 1023 bp Std Dev 66. 3 bp CV 6. 5% Proprietary 23

Setting up Samples in 15 µl Sample Tubes Why centrifugation ? • Dynamic DNA mass range of 1 µg to 10 µg • Mate Pair libraries • Raindance library prep Proprietary 24

Preparing the M 220 for Use • Turn on M 220 and start computer • Open Sono. Lab software • Fill water bath with the XTU holder in place with AFA-grade water • Ensure Water Level is within specified operating levels • Select a Method in the Method box - this will set the run parameters • Protocol will initiate once the water temperature reaches specified range • Load sample vessel into the XTU tube holder and slide the weight into the running position • Close the safety enclosure door • Select Run Proprietary 25

Preparing the M 220 for Use Draining water and cleaning • Special care needs to be taken when draining the water from water bath. • Remove the tube holder and place it on a clean, dry surface. Draw the water from the acoustic assembly using the provided syringe. It is important to use the plastic tip on the syringe to prevent the scratch of the transducer surface. Water needs to be drawn as completely as possible. • Use the disposable cleaning swab to sweep the water channels in the Acoustic Assembly. Wipe the surfaces of the transducer and the metal housing with lint-free cloth and air dry for next use. • Store the Tube Holder Insert in a dry place Shutdown Procedure • If the instrument will be idle for an extended time (e. g. overnight or a weekend), shut down the instrument using the following procedure: Empty the water bath, store the Tube Holder Insert in a dry place. Proprietary 26

Additional Applications • tru. Ch. IP® Chromatin Shearing Kits • • tru. XTRAC® FFPE t. NA, DNA, and RNA Kits • • Sample Prep for Mycobacterium spp. Protein Extraction Buffers • • Single-tube blood collection, stabilization, transport, and biomarker extraction solution tru. XTRAC MALDI-TOF • • Improved DNA yields from Dried Blood Spots tru. COLLECT™ • • NGS-grade RNA and DNA Extraction from FFPE tissues tru. XTRAC DBS • • Optimized reagents and protocols for chromatin shearing with AFA Focused-ultrasonicators A range of extraction buffers for the efficient protein extraction from cells and tissues optimized for your downstream application DNA Shearing Verification Kit • Verify the performance of your AFA Focused-ultrasonicators with the DNA Shearing Verification Kit Proprietary 27

Contact – United States US HEADQUARTERS Covaris, Inc. 14 Gill Street, Unit H Woburn, Massachusetts 01801 -1721 USA Tel: +1 781 -932 -3959 Fax: +1 781 -932 -8705 www. covaris. com If you need further assistance our staff of Application Scientists, Engineers, and Technical Specialists are committed to providing you with prompt and professional support. For service and support for your Covaris instruments and consumables in the US or Europe, contact: Tech. Support@covaris. com For Application Support in the US or Europe, contact: Application. Support@covaris. com. To Place Orders in the US, contact: Customer. Service@covaris. com. Proprietary 28

Contact – International Europe, Middle East, and Africa Covaris, Ltd Unit 3, Brighton Office Campus Hunns Mere Way Woodingdean Brighton BN 2 6 AH United Kingdom Tel: +44 (0)845 872 0100 Fax: +44 (0) 845 384 9160 To Place Orders in Europe, contact: EMEACustomer. Service@covaris. com www. covaris. com International Our international distributors provide Covaris products to laboratories around the world. For information about international product distribution opportunities, please contact International. Sales@covaris. com Proprietary 29