diagenode Bioruptor Pico User manual

USER MANUAL
Version 1.1
Bioruptor® Pico
Cat. No. B01060001
Bioruptor® Pico
Sonication System

Guarantee
Limited one year global warranty
Diagenode guarantees all products from any manufacturing defects as we rigorously test all products to meet strict
quality standards. Diagenode warrants that all standard components of its instruments will be free of defects in materials
and workmanship for a period of one (1) year from the date that the warranty period begins, unless the original quotation
or accompanying documentation states a different warranty period. All warranty periods begin on the date of delivery
and apply only to the first purchaser of the product. If a manufacturing defect arises and a valid claim is received within
the warranty period, Diagenode, at its discretion, will repair or replace the product in accordance with the warranty
terms and conditions stated herein. In case of repair or replacement of a product under warranty, Diagenode will cover
the expenses to return the repaired or replacement product.
This warranty covers only manufacturing defects and does not cover any damage caused by misuse, lack of compliance
to recommendations stated in the manual, neglect, accidents, abrasion, or exposure to extreme temperatures, chemical
solvents, or acids. We strongly recommend that maintenance or repairs of Diagenode’s products are performed by our
approved Diagenode service center. Improper or incorrectly performed maintenance or repairs will void the warranty.
Technical Assistance & Ordering Information
Diagenode s.a. BELGIUM | EUROPE
Avenue de l’hôpital,1
Tour GIGA, 3rd Floor
4000 Liège - Belgium
Tel: [phone]
Fax: [phone]
[e-mail]
[e-mail]
Diagenode Inc. USA | NORTH AMERICA
400 Morris Avenue, Suite #101
Denville, NJ 07834 - USA
Tel: [phone]
Fax: [phone]
[e-mail]
[e-mail]
For a complete listing of Diagenode’s international distributors, visit:
http://www.diagenode.com/en/support/distributors.php
For the rest of the world, please contact Diagenode s.a.

PAGE 3
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Critical Steps for Maintenance and Efficient Shearing ....................................4
Introduction .........................................................................5
Bioruptor® Pico Technical Specifications ................................................6
Getting to Know Your Bioruptor® Pico System ............................................7
Bioruptor® Pico Components Overview ...............................................7
Bioruptor® Pico Water Cooler .......................................................7
Single Cycle Valve for Bioruptor® Pico ................................................8
Sonication bath ...................................................................8
Motorized lid .....................................................................9
Tube holders .....................................................................9
Equipment Installation ..............................................................10
Installation overview .............................................................10
Installing the Bioruptor® Pico System with Water Cooler ................................11
Controlling the Sonication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Tube Holders and tubes ..............................................................16
Standard protocol for DNA Shearing ...................................................17
Operating Conditions .............................................................17
Important comments about DNA shearing ...........................................20
Small Volume DNA Shearing for Bioruptor® Pico ........................................22
Operating Conditions .............................................................22
Important comments about DNA shearing ...........................................25
Consistent and highly reproducible chromatin shearing with the Bioruptor® Pico ...........25
Ordering Information ........................................................Back Cover
Contents

PAGE 4 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Critical Steps for Maintenance and
Efficient Shearing
General warnings
• DO NOT turn on the instrument without water.
• DO NOT tilt the Bioruptor® Pico. To change the water, use either the plastic pump or a beaker (be careful not to
scratch the bottom of the sonicaton bath).
Sonication bath levels
• The sonication bath must be filled with distilled water to the fill line. Fill line replacement stickers can be obtained by
contacting Diagenode.
• Change water at least once per week.
Sonication bath temperature
• Optimal temperature for sonication is 4°C. Sample temperature should not exceed 8°C.
• The Water Cooler (Cat. No. BioAcc-Cool) has to be used in combination with the Single Cycle Valve for Water Cooler
(Cat. No. B02020004) to guarantee the automatic temperature control of the sonication bath during the entire
sonication process.
Magnetic Ultrasound Emitter Maintenance
• The ultrasound waves are created from a series of magnets that are attached to the water tank. This system is very
sensitive to the heat generated during a run.
• Do not exceed 1 hour of total sonication per run. It is critical that the machine is allowed to cool at least 20 minutes
between runs. Damage resulting from non compliance to manual instructions will void the warranty and shorten the
lifespan of the machine.
• Ultrasound emitters can be damaged by tilting or jarring the machine. Exercise care if moving water tank.
Validated tubes for the Bioruptor® Pico
• DNA shearing: 0.1 ml (Cat.No. C30010015) and 0.65 ml (Cat.No. WA-005-0500) Bioruptor® Microtubes for DNA
shearing.
• Chromatin shearing: 0.1 ml (Cat. No. C30010015) and 1.5 ml (Cat.No. C30010016) Bioruptor® Microtubes and 15 ml
(Cat.No. C01020031) Bioruptor® Tubes & sonication beads.
Fitting 0.1, 0.65, 1.5, or 15 ml tubes in the tube holder
1. Place the tubes in the corresponding tube holder (0.1 ml tube holder & tube adaptor - Cat. No. B01200041,
0.65 ml tube holder – Cat. No. B01200042, 1.5 ml tube holder - Cat. No. B01200040 or 15 ml sonication accessories
- Cat. No. B01200016). Never leave empty spaces in the tube holder. Fill the empty spaces with tubes containing the
same volume of water. Screw the lid on the tube holder without over-tightening it.
2. Carefully place the tube holder on the holding plate.
3. During sonication, samples must remain at the bottom of the tube. If needed, briefly centrifuge samples during
sonication after pausing the run.
Bioruptor

PAGE 5
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Introduction
Diagenode’s Bioruptor® Pico uses a gentle method of sonication to retain the integrity of DNA and/or biological complexes,
including chromatin, protein-protein binding, protein-DNA complexes and other biochemical and biological assay systems.
The Bioruptor® Pico sonication system uses a sonication bath to generate indirect sonication waves, which emanate from an
ultrasound element below the water tank. Because the system is gentler than other sonicators, the Bioruptor® Pico produces
better and more consistent results than with harsher sonication methods. Up to 12 closed tubes can be sonicated in parallel
and the continuous rotation of tubes allows even distribution of the energy for efficient sonication. The Bioruptor® Pico enables
automation of sonication, guaranteeing higher reproducibility of results.
The effect of ultrasound on biological samples.
The Bioruptor® Pico sonication system uses ultrasound to create
focused mechanical stress to shear chromatin or DNA. Ultrasound
waves pass through the sample expanding and contracting the liquid.
During expansion, negative pressures pull the molecules away from
one another to form a cavity or bubble. This process is called cavitation.
The bubble continues to absorb energy until it can no longer sustain
itself and implodes. This produces intense focused shearing forces,
that disperse and break biomolecules. The fragmentation of chromatin
or DNA takes place as a consequence of this mechanical stress or
shear.
With the Bioruptor® Pico, the entire volume of water present in the sonication bath is exposed to ultrasound, allowing all
the samples to be efficiently sonicated in parallel (Figure 1).
Use of Bioruptor® Pico by pregnant women
Exposure to 20-60 kHz sound waves has not been shown to be harmful to human health. However, we would recommend
avoiding unnecessary exposure. Diagenode recommends that pregnant women should not be exposed to 20-60 kHz wave
lengths for a long period of time.
Fig 1. Propagation in 0.65 ml tubes
Ultrasound
Transducer

PAGE 6 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Bioruptor® Pico Technical Specifications
Bioruptor® Pico
Power Supply 100-230V, 50/60Hz, 2.1A (EU) / 4.2A (US)
Ultrasonic wave frequency 20-60 kHz
Ultrasonic wave output power 25-210 W
Sonication unit dimensions 350 (W) x 245 (D) x 280 (H) mm
Sonication bath volume 750 ml
Timer Digital
Possibility to control water flow via connector kit for water cooler
Yes
Tube holder Available for 0.1, 0.65, 1.5 or 15 ml tubes
Total weight 7,90 kg
Number of samples to be processed simultaneously
0.1 ml tubes – 12
0.65 ml tubes – 12
1.5 ml tubes – 6
15 ml tubes – 6

PAGE 7
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Getting to Know Your Bioruptor® Pico System
Bioruptor® Pico Components Overview
Water Cooler
To learn more, please visit www.diagenode.com
Power Cable (EU) Power Cable (US)
Sonication Unit
Sonication bath
Motorized lid
Tube holder

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Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Single Cycle Valve for Bioruptor® Pico
To learn more, please visit www.diagenode.com
Sonication bath
The sonication bath is a critical component of the instrument. The generators below the tank produce ultrasonic waves
which are then transferred through water. The sonication bath requires special handling and care as described below.
Handling
The sonication unit must remain upright at all times, especially when moved. Tilting the sonication unit or handling
roughly may damage the ultrasound emitter component, resulting in a substantial drop in sonication efficiency. If
transportation of the apparatus is required after initial set-up, it is imperative to keep the sonication unit at a right angle
to the ground during the transport at all times.
Water level and quality
The level of the water has been optimized and should always reach the red line (sticker on the wall of the tank). Distilled
water should be used to fill the tank. Replacement stickers can be obtained from Diagenode.
Water temperature
The water in the sonication bath must be kept at 4°C. Ultrasonic waves produced by the Bioruptor® Pico generate heat.
Drop off in sonication efficiency will occur above 8°C. To ensure preservation of the samples and to prevent damage to
the instrument, it is necessary to start the sonication process with cold water and to keep it at 4°C during the sonication
process.
Single Cycle Valve Tubing Connector cable

PAGE 9
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Automatic temperature control
The Water Cooler (Cat. No. BioAcc-Cool) has to be used in combination with the Single Cycle Valve for Bioruptor® Pico
(Cat. No. B02020004) to guarantee the automatic temperature control of the sonication bath during the entire sonication
process (Figure 2). The cooling system features two pumps and produces a regular water flow to maintain a constant
water level in the tank. The regulating valve (Single Cycle Valve for Bioruptor® Pico) ensures that water will only be
replaced during the off cycle to avoid any interference between the water flow and the sonication process.
Note: You may permanently install the Bioruptor® Pico in a cold room, though this is not sufficient to avoid the temperature
increase during sonication.
Motorized Lid
The motorized lid, along with the gear plate accessory, keeps the sample tubes in constant
rotation and ensures optimal position in the sonication bath during sonication. When in motion,
do not hamper the rotation of the white gear plate. Avoid the immersion of the motor into the
water. Do not heat the white plastic as it will warp.
Tube Holders
Several sizes of tubes can be used with the Bioruptor® Pico.
The minimum and maximum sample volume to be used with each tube is given in the table below.
Tube size Minimum Maximum
0.1 ml 5 µl 10 or 50 µl
0.65 ml 100 µl 100 µl
1.5 ml 200 µl 300 µl
15 ml 500 µl 2 ml
Water Cooler
Sonication unit
Single Cycle Valve
Fig 2. Setup of the Bioruptor® Pico system including Water Cooler and Single Cycle Valve for Bioruptor® Pico.

PAGE 10 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Equipment Installation
The following pages contain information on installing your particular Bioruptor® Pico model. This equipment must only
be installed by personnel after reading this section. Consider all hazards even though no particular hazards have been
identified during installation. Before starting installation work, turn the main switch off (front side) and secure the unit
against being re-energized. No special tools are required. One square meter is needed to set-up the Bioruptor® Pico.
Devices are designed to be safe under the following conditions:
Installation overview
• Indoor use
• Altitude up to 2,000 meters
• Operating external temperature 0°C to 25°C
• Maximum relative humidity 80%
• Transient overvoltage typically present on the
MAINS supply
• Degree of protection: IP20
• Power plug must be grounded
• POLLUTION DEGREE 2 (Normally only non-conductive
pollution occurs. However, occasionally a temporary
conductivity caused by condensation is expected)
• Never install this equipment in a place where
environmental conditions and warnings mentioned
above are infringed
Power cord
Distilled water
Digital timer
Water Cooler
Power cord
Motorized lid
Tube holder
Connector kit for
Water Cooler
Tubing
Bioruptor® Pico
Fig 4. Schematic installation overview of the Bioruptor® Pico System
in combination with the Water Cooler and the Single Cycle Valve kit.

PAGE 11
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Installing the Bioruptor® Pico System with Water Cooler
Before starting the installation, turn the main switches off and make sure that the unit is not plugged into an electrical
outlet.
1. Open the boxes and unpack all components.
2. Place the Bioruptor® on a bench.
3. Place the Water cooler below the Bioruptor®.
4. Place the Single Cycle Valve on top of the Water cooler as shown in the image.
Location Requirements: The Water cooler must be located below the Bioruptor® Pico (minimum
elevation difference 400 mm or 15,74 inch).
Important Note: Please make sure that the Bioruptor® Pico is always placed on a level surface.
Water cooler
(BioAcc-cool)
Bioruptor® Pico
(B01060001)
Single Cycle Valve
(B02020004)

PAGE 12 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
5. Insert the short red and blue tubing into the outlets of the Water cooler and the lower red and blue nozzle connectors
( ) of the single cylce vavle box as shown in the image. Respect the red and blue color codes.
7. Plug the Single Cycle Valve cable into the outlet on the back side of the Single Cycle Valve Box and on the Bioruptor®
Pico.
8. Plug the power cord into the outlet and switch on the power switch on the back side of the sonication unit.
8. Plug the power cord into the outlet of the Water cooler.
6. Connect the Bioruptor® Pico to the Water cooler with the long red and blue tubing by inserting them into the upper
red and blue nozzle connectors ( ) of the single cycle valve box as shown in the image. Respect the red and blue color
codes. (Optional: Cut the length you need for the output and input flow. Make sure there is enough slack.)
Important Note: If you need to cut a part of the tubing, always use the provided Cutting Device. Never
use scissors because pieces of tubing with bad cuts generate leaks at junctions. Only properly cut
tubes can be inserted in the connectors.
(Note: Red connector and red tubing will carry water from the sonication bath to the Water cooler.
Blue connector and blue tubing will carry water from the Water cooler to the sonication bath).

PAGE 13
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
11. Fill the tank of the Water cooler with 3.5 liter and the sonication bath of the Bioruptor® up to the red line of
the sticker (700 or 730 ml; depending on the model) with distilled water only (do not use deionized water!).
11. Temperature can be set to 4°C by pushing the SET key and the arrow key of the control panel
at the same time..
12. Press Start/Stop key to control the temperature.
Note: See Water cooler manual for additional information
Now you are ready to start !
10. Press main switch on the front side of the Water cooler.
Main Switch

PAGE 14 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Controlling the Sonication
1Digital Timer:
Allows the user to easily program the sonication of samples (ON/OFF pulse time & total time). See use of
digital timer in next section below.
2Start Button (A):
Begins sonication
3Stop Button (B):
Cancels sonication
Switch on power switch (back side of sonication unit)
1
2
3

PAGE 15
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
CYCLE NUMBER, TIME ON and TIME OFF are the
parameters controlling sonication. For changing these
parameters press the OK button. First the TIME ON
screen appears with the flashing number. This parameter
can be modified with the up ( ) and down button ( ).
Please respect the range for each value. Press again the
OK button to confirm the setting. To select TIME OFF and
Cycle Num proceed the same way.
Bioruptor® Pico will sonicate as shown.
Once parameters are set press START (Figure 2).
The information: Close the lid to start the run indicates
that the sonication only starts when the acrylic glass
cover is closed. The cover also prevents from any noise
disturbance.
Once the run started the RUNNING screen appears. As
shown in Figure 4 the RUNNING screen indicates the
elapsed time ON, time OFF and the number of cycles.
If the acrylic glass cover will be opened during a run the
sonication will automatically pause. Once the cover is again
closed the machine continues with the sonication process.
As shown in Figure 5, the Break Cycle screen shows the
information when the run stopped. In the example (Figure
5) the machine stopped during the second sonication
cycle, after 30 sec of TIME ON and 8 sec of TIME OFF.
If the sonication process worked properly (without any
error messages) the RUN OVER screen appears (Figure 6).
Press ESC to get back to the Main screen to start another
sonication round.
Digital Timer
Figure 1. Main screen
Time ON 30sec
Time OFF 30sec
Cycle Num 10
OK = change params
ON
OFF
TIME
ON
TIME
OFF
CYCLE
START
Cycle number reached
(4 cycles in this example)
IMPORTANT NOTE: Machine has to rest for 20
minutes before starting the next sonication run.
Time ON 30sec
Time OFF 30sec
Cycle Num 10
Press START to go
Close the lid to
start the run
OR
Press STOP
ON 07s
OFF 00s 01/10
RUNNING
Break cycle: 02
T 2: 08
Close the Cover to
continue or STOP
T 1: 30
Figure 2. Main screen
Figure 4. RUNNING Screen
Figure 3. Close Lid Screen
Figure 5. Break
Press ESC to go
back to main menu
RUN OVER
Figure 6. Run over
Figure 1. Main screen

PAGE 16 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Tube holders & tubes
For chromatin and DNA shearing we highly recommend to use the below mentioned tube holders and the
corresponding tubes.
To guarantee homogeneity of chromatin or DNA shearing, the tube holders should always be completely filled with
tubes. Never leave empty spaces in the tube holder. Fill the empty spaces with tubes containing the same volume of
distilled water.
1.5 ml tube holder for
Bioruptor® Pico
(Cat. No. B01200040)
15 ml sonication accessories
for Bioruptor
®
Pico
(Cat. No. B01200016 )
15 ml Bioruptor® tubes
& sonication beads
(recommended
for chromatin, not
recommended for DNA
shearing)
(Cat. No. C01020031)
0.65 ml tube holder for
Bioruptor® Pico
(Cat. No. B01200042)
0.1 ml tube holder & tube
adaptor for Bioruptor® Pico
(Cat. No. B01200041)
0.65 ml Bioruptor®
Microtubes (recommended
for chromatin & DNA
shearing)
(Cat. No. WA-005-0500)
1.5 ml Bioruptor®
Microtubes (recommended
for chromatin, not
recommended for DNA
shearing)
(Cat. No. C30010016)
0.1 ml Bioruptor®
Microtubes (recommended
for chromatin & DNA
shearing)
(Cat. No. C30010015)

PAGE 17
DIAGENODE BIORUPTOR® PICO USER MANUAL
Diagenode Inc. North America / Phone: [phone] // Fax: [phone] // Mail: [e-mail]
Standard protocols for DNA shearing
Standard operating conditions
Sample volume: 100 µl
Tubes: 0.65 ml Bioruptor® Microtubes (Cat. No. WA-005-0500)
Tube holder: 0.65 ml tube holder for Bioruptor® Pico (Cat. No. B01200042) for 12 x 0.65 ml tubes
Sonication buffer: TE (10 mM Tris, 1mM EDTA, pH 7.5 - 8.0)
DNA concentration: 1-20 ng/µl (10 ng/µl recommended)
Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing.
For optimal results samples should be stored on ice during 5-10 minutes prior to sonication.
Temperature: 4°C – Water Cooler (Cat. No. BioAcc-Cool) & Single Cycle Valve (Cat. No. B02020004)
Sonication cycle & total sonication time: varies depending on desired DNA size (see table)
Note: Recommended protocols are subject to change without notice. Additional protocols are available on demand.
125 bp
135 bp
183 bp
200 bp
234 bp
319 bp
476 bp
696 bp
848 bp
1036 bp
1230 bp
Programmable DNA size distributions,
excellent reproducibility, and high dsDNA
yields with Bioruptor® Pico
Figure shows different DNA size distributions
of sheared genomic DNA produced by varying
the duration of sonication. The different
curves depict a specific Bioruptor® Pico
Next-Generation run, optimized to produce
specific mean sizes and size ranges for Next-
Generation sequencing.
All samples were analyzed on Bioanalyzer 2100
using DNA High Sensitivity chip.

PAGE 18 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Target size Cycle condition
(On/Off cycle time) Cycle number
150 bp 30’’/30’’ 30
200 bp 30’’/30’’ 13
300 bp* 30’’/90’’ 6
400 bp* 15’’/90’’ 7 - 8
1000 bp* 5’’/90’’ 7 - 8
* For longer fragments (300 up to 1000 bp), a short centrifugation step after half of the cycle numbers can significantly improve the results.
Protocols for other size ranges (incl. longer fragments up to 1300 bp) are available on request.
Broad DNA size distribution, excellent
reproducibility, and high dsDNA yields with
Bioruptor
®
Pico
Figure A shows different DNA size distributions
of sheared genomic DNA produced by varying
the duration of sonication from 2 separate
experiments (run-to-run variation). The different
lanes depict 4 lanes from 2 different Bioruptor
®
Pico runs, optimized to produce specific mean
sizes corresponding to 150, 200, 300 and 400
bp (based on the standard Ion Torrent standard
sample preparation size requirements).
Figure B shows different DNA size distributions
of sheared genomic DNA produced by varying the
duration of sonication. The different lanes depict a
specific Bioruptor
®
Pico run, optimized to produce
specific mean sizes and size ranges for Next-
Generation DNA sequencing.
Panel A: peak electropherogram view. Lanes 1,2:
150 bp; Lanes 3,4: 200 bp; Lanes 5,6: 300 bp; Lanes
7,8: 400 bp.
Panel B: gel virtual view
All samples were analyzed on Bioanalyzer 2100
using DNA High Sensitivity chip.
A.
B.
The protocol settings listed above are recommended guidelines and actual results may vary depending on the type and amount
of starting material, purity level, concentration and/or sample viscosity. It is highly recommended that a time course response
experiment be carried out (e.g. varying the time of “on” and “off” durations as well as the number of cycles) to determine the
appropriate treatment for your specific sample. Starting material with a smaller sample volume and a greater concentration
than the recommended range may require a different time course to ensure homogenous shearing results.

PAGE 19
DIAGENODE BIORUPTOR® PICO USER MANUAL
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A. Target Size: 150 bp
C. Target Size: 300 bp
B. Target Size: 200 bp
D. Target Size: 400 bp
Excellent reproducibility and programmable
size distribution with Bioruptor
®
Pico
Panels A to D show size distributions of
sheared genomic DNA from separate
experiments aiming at producing high-quality
DNA fragments compatible with Ion Torrent
standard sample preparation protocols (150,
200, 300 and 400 bp).
Panel A: 30 cycles, 30 sec ON/30 sec
OFF cycles. Expected fragment size: 150
bp, observed average fragment size is
150.6 bp (CV%: 2.07%).
Panel B: 13 cycles, 30 sec ON/30 sec
OFF cycles. Expected fragment size: 200
bp, observed average fragment size is
202.2 bp (CV%: 4.7%).
Panel C: 6 cycles, 15 sec ON/90 sec OFF
cycles. Expected fragment size: 300
bp, observed average fragment size is
291.3 bp (CV%: 4.31%).
Panel D: 7 cycles, 15 sec ON/90 sec OFF
cycles. Expected fragment size: 400
bp, observed average fragment size is
406.9 bp (CV%: 7.96%).
All samples were analyzed on Bioanalyzer
2100 DNA High Sensitivity chips.

PAGE 20 DIAGENODE BIORUPTOR® PICO USER MANUAL
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: [phone] // Mail: [e-mail]
Important comments about DNA shearing
The Diagenode ACT (Adaptative Cavitation Transfer technology) process is highly reproducible. However, attention must
be paid to the following treatment attributes to ensure best results:
• Tubes: At present, the recommended tube vessels are the 0.65 ml Bioruptor® Microtubes (Cat No. WA-005-0500). Pay
attention not to damage the cap when closing the tubes since this could alter sonication results.
• Sample volume: The recommended volume of the 0.65 ml Bioruptor® Microtubes (Cat No. WA-005-0500) is 100
µl. When using lower volumes (e.g. ≤ 50 µl), less reproducible results may be observed due to an alteration of the
ultrasonic waves distribution in the sample fluid; thus, reducing the efficiency of sonication which may result in
broader size distribution or larger peaks.
• Sample concentration: Diagenode recommends using DNA concentration ranging between 1 and
20 ng/µl (10 ng/µl recommended). Using larger concentration (e.g. 50-100 ng/µl) may result in broader peaks or
variable peak distribution.
• Sample preparation: Sample viscosity may have a major impact on sonication results. Careful resuspension of DNA
sample is strongly recommended before sonication processing. Multiple pipetting and gentle vortexing followed by a
short centrifugation to recover sample volume at the bottom of the tube is therefore strongly recommended. Storing
DNA samples on ice during 5-10 minutes before sonication has also been shown to improve reproducibility.
• DNA quality: DNA quality and quantity must be considered carefully since bad quality and quantity DNA may have
several impacts on sonication and Next-Gen sequencing downstream applications. First, DNA contamination
(e.g. from superfluous nucleic acids such as RNA, small nucleic acid fragments, excess proteins, or other
contaminating materials) may interfere with DNA measurement method leading to incorrect DNA quantitation thus.
Also contaminating RNA in genomic DNA preparation might generate a biased fragment distribution profile on
microfluidics-based platform (e.g. Agilent Bioanalyzer) or alter sonication effiency.
Therefore it is highly recommended to use only high quality DNA when sonicating DNA for Next-Gen sequencing
library preparation. The DNA sample to be processed should be highly pure, having an OD260/280 ratio of between 1.8
and 2.0, and should be as intact as possible. DNA extracted using standard techniques (e.g. Proteinase K digested,
double phenol/chloform extraction, ethanol precipitated, treatment with RNase-DNase free enzymatic digestion to
remove contaminant RNA) or commercial spin-column based kits are recommended.
• Water temperature: Propagation of ultrasound in a liquid unavoidably produces heat that can ultimately alter DNA
sample (e.g. by thermal denaturation). To ensure the best preservation of the sample, it is recommended to start the
sonication process with cold water in the water bath. During sonication, especially when doing long sonication runs,
the temperature must also be controlled. This is obtained by the automatic temperature control.
Note: The permanent installation of the Bioruptor® Pico in a cold room is possible, although not sufficient to avoid the
temperature increase due to sonication.
• Automatic temperature control: A recirculating water cooler is used to guarantee the automatic temperature
control of the water bath during the whole sonication process. This water cooler (Cat. No. BioAcc-cool) produces a
regular water flow with a constant water level in the tank. An additional regulating valve (Single Cycle Valve, Cat. No.
B02020004) ensures that water will only be replaced during the off cycle to avoid any interference between the water
flow and the sonication process.
• Sonication time: Minor adjustments in cycle number may be made to optimize results for various sample types and
concentrations. Cycle number listed above is a recommended guideline. Actual results may vary depending on the
amount and type of starting material, concentration, viscosity and/or plastic tubes. Diagenode recommends setting
up a time dose response experiment for determining appropriate cycle number. Larger length starting material (e.g.
total genomic DNA) and higher concentration may require a longer dose to ensure a homogeneous shearing result.
• Water bath: The sonication water bath is a critical component of the Bioruptor® Pico sonication system.