- Diagenode
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1. 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 New Cat No B0201002 230V B02010003 115V B02010004 100V Old 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 soni2. 4 Press ESC to get back to the Main screen to start another sonication round Figure 2 Main Screen 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 Figure 6 RUN OVER Screen IMPORTANT NOTE Machine has to rest for 20 minutes before starting the next sonication run Figure 3 Close Lid Screen www diagenode com diagendtie PAGE 16 DIAGENODE BIORUPTOR PICO USER MANUAL 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 RA i o c 0 1 ml tube holder amp tube 0 65 ml tube holder for 1 5 ml tube holder for 15 ml sonication accessories adaptor for Bioruptor Pico Bioruptor Pico Bioruptor Pico for Bioruptor Pico Cat No B01200041 Cat No B01200043 Cat No B01200040 Cat No B01200016 0 1 ml Bioruptor 0 65 ml Bioruptor 1 5 ml Bioruptor 15 ml Bioruptor Tubes Microtubes recommended Microtubes recommended Microtubes recommended recommended for Tissue for chromatin amp DNA for chromatin amp DNA for chromatin not disruption for protein sh
3. 500 000 cells are analyzed on a 296 agarose gel Note For shearing in a final volume of 500 ul or 1 ml the 15 ml Bioruptor Tubes are filled with sonication beads up to the first or second line of the graduation scale 0 1 or 0 2 ml corresponding to around 250 or 450 mg of beads 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 27 www diagenode com diagendtie Ordering information diageng e Innovating Epigenetic Solutions Products Bioruptor Models Cat No Bioruptor Pico sonication device for 0 65 ml tube holder B01060001 Bioruptor Pico sonication device for 0 1 amp 0 65 ml tube holder B01060002 Bioruptor Pico sonication device for 1 5 ml tube holder B01060003 Cooling System Single Cycle Valve for Bioruptor Pico B02010002 Consumables 0 65 ml tube holder for Bioruptor Pico B01200043 1 5 ml tube holder for Bioruptor Pico B01200040 15 m
4. gt 100 gt 100 gt A 10 ul volume B 100 pl volume C 300 pl volume Panel A 10 ul volume Chromatin samples are sheared for 10 15 and 20 cycles of 30 sec ON 30 sec OFF with the Bioruptor Pico using 0 1 ml Bioruptor Microtubes Cat No C30010015 Panel B 100 ul volume Chromatin samples are sheared for 10 cycles of 30 sec ON 30 sec OFF with the Bioruptor Pico using 0 65 ml Bioruptor Microtubes New Cat No C30010011 Old Cat No WA 005 0500 Panel C 300 ul volume Chromatin samples are sheared for 5 10 and 15 cycles of 30 sec ON 30 sec OFF with the Bioruptor Pico using using 1 5 ml Bioruptor Microtubes Cat No C30010016 Prior to de crosslinking samples are treated with RNase cocktail mixture at 37 C during 1 hour The sheared chromatin is then de crosslinked overnight and phenol chloroform purified as described in the kit manual 10 ul of DNA equivalent of 500 000 cells are analyzed on a 2 agarose gel MW corresponds to the 100 bp DNA molecular weight marker MW corresponds to the 100 bp DNA molecular weight marker 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 treat
5. 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 diagencHe Single cycle valve Water Cooler Sonication unit Fig Setup of the Bioruptor Pico system including Water Cooler and Single Cycle Valve for Bioruptor Pico 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 o pl 10 or 50 ul 0 65 ml 100 ul 100 ul 1 5 ml 200 ul 300 ul tomi 900 ul Aml www diagenode com diagendue PAGE 10 DIAGENODE BIORUPTOR PICO USER MANUAL Equipment installation The following pages contain information on installing your particular Bioruptor Pico model This equipment must only be installed by personnel
6. sonication bath must be filled with distilled water to the fill line Fill line replacement stickers can be obtained by contacting Diagenode e Change water at least once per week Sonication bath temperature e Optimal temperature for sonication is 4 C Sample temperature should not exceed 8 C e The Water Cooler New Cat No B0201002 230V B02010003 115V B02010004 100V Old 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 e 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 e 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 e Ultrasound emitters can be damaged by tilting or jarring the machine Exercise care if moving water tank Validated tubes for the Bioruptor Pico e DNA shearing 0 1 ml Cat No C30010015 and 0 65 ml New Cat No C30010011 Old Cat No WA 005 0500 Bioruptor Microtubes for DNA shearing e Chromatin shearing 0 1 ml Cat No C30010015 and 1 5 ml Cat No C30010016 Biorupto
7. water bath during the whole sonication process This water cooler New Cat No B0201002 230V B02010003 115V B02010004 100V Old 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 Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 21 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation resulting in broader size distribution or larger peaks Bath water should be pure distilled water changed regularly at least once per week 2 Water bath maintenance he water bath
8. 6 og eee ees 7 Eg ipmeni installation 4339 23 94 94 9 35 9 inara dod ee ac aa oe aa ee ea 10 Ipxcd uEo d MMRMET 10 Installing the Bioruptor Pico system with Water Cooler llis 11 Controlling the SOnICallOEk s sx ERES UE RES DEEL ERE ER ESG be eee Ses Bee Rd LENT ED EP dig Es 14 Tube holders and tUDS5S s 3derpoo 13i don dubi qe dioi iw dp aree d ibid dd bred indian decd 16 Standard protocol for DNA shearing lllllllllleeeee hh hh 17 BlerisciuineRee gelo PL P 17 Important comments about DNA shearing 0 000 es 20 Small volume DNA shearing for Bioruptor Pico 000 cece eee eee ee 22 Opera imo CORI Oe aa i wae are aaa det a ee wee ee ey ee od ee tay E a aes 22 Important comments about DNA shearing axes siei acei maa aae a aaa aaa i ay aA a E a a e ara 25 Consistent and highly reproducible chromatin shearing with the Bioruptor Pico 20 Ordering informatio sedrs ssas ta Gor Groom etai onus Ros Boom Kee o Oase ewe as doo pus dod Back Cover PAGE 3 www diagenode com diagenctie PAGE 4 Innovating DIAGENODE BIORUPTOR PICO USER MANUAL General warnings N e DO NOT turn on the instrument without water e 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 e he
9. R PICO USER MANUAL 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 Wes cooler Tube holder Available for 0 1 0 65 1 5 or 15 ml tubes Total weight 7 90 kg 0 1 ml tubes 12 0 65 ml tubes 12 1 5 ml tubes 6 15 ml tubes 6 Number of samples to be processed simultaneously DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 7 Getting to know your Bioruptor Pico system Bioruptor Pico components overview Sonication bath Motorized lid Sonication unit ef Power cable EU Power cable US Tube holder Water Cooler To learn more please visit www diagenode com www diagenode com diagendtie PAGE 8 DIAGENODE BIORUPTOR PICO USER MANUAL Single cycle valve for Bioruptor Pico Single cycle Valve Tubing Connector cable 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 handl
10. Recommended protocols are subject to change without notice Additional protocols are available on demand Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL Sonication table for small volume DNA Shearing Cycle conditions On Off time Cycle number 200 bp 30 30 20 cycles 250 bp 307 30 15 cycles 300 bp 307 30 13 cycles 550 600 bp 157 90 7 cycles 750 800 bp 157 90 6 cycles Short centrifugation after 5 and 10 cycles added Short centrifugation after 4 and 8 cycles added Short centrifugation after 3 cycles added e 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 or a greater concentration than the recommended range may require a different time course to ensure homogenous shearing results e Adding short centrifugation step s during the sonication round e g after half of the cycle numbers can significantly improve the results e Protocols for other size ranges are available on request Small volume DNA shearing results Programmable DNA size distribution and high reprod
11. 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 e Indoor use e Power plug must be grounded e Altitude up to 2 000 meters e POLLUTION DEGREE 2 Normally only non conductive pollution occurs However occasionally a temporary e Operating external temperature 0 C to 25 C ia uid conductivity caused by condensation is expected e Maximum relative humidity 80 e Never install this equipment in a place where environmental conditions and warnings mentioned above are infringed e Transient overvoltage typically present on the MAINS supply e Degree of protection IP20 Installation overview Tube holder Motorized lid Distilled water Connector kit for Water Cooler Single cycle diagencde valve Power cord SS a SS BIORUPTOR PICO diagenctle minichiller Power cord WATER COOLER Fig Schematic installation overview of the Bioruptor Pico System in combination with the Water Cooler and the Single Cycle Valve kit Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 11 Installing the Bioruptor Pico system with Water Coo
12. ate 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 8 Plug the power cord into the outlet of the Water cooler Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 13 10 Press main switch on the front side of the Water cooler Main Switch 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 5 amp 1 and the arrow key of the control panel at the same time i Display LED display status unc WL Araw kavel SET key Start stop key 12 Press Start Stop key to control the temperature Note See Water cooler manual for additional information Now you are ready to start www diagenode com diagendue PAGE 14 DIAGENODE BIORUPTOR PICO USER MANUAL Controlling the sonication 1 Digital Timer Allows the user to easily program the sonication of samples ON OFF pulse time amp total time See use of digital timer in next section below 2 Start Button Begins sonication Stop Button Cancels sonication Switch on po
13. cation 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 e Water bath The sonication water bath is a critical component of the Bioruptor Pico sonication system 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation resulting in Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 25 broader size distribution or larger peaks Bath water should be pure distilled water changed regularly at least once per week 2 Water bath maintenance he water bath metal surface is fragile and requires a careful maintenance Use only soft sponge to remove traces Never use scratch scrub sponge since this would alter the ultrasonic wave emitter surface 3 Water type Distilled water Supplementary Data Please note that there are three main sources of variation in both peak base pair size and distribution 1 The physical process of DNA fragmentation might not be entire
14. diageng e Innovating Epigenetic Solutions USER MANUAL Bioruptor Pico 5onication System Cat No B01060001 B01060002 B01060003 Version 1 21 09 15 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 Dia
15. e volume The recommended volume of the 0 65 ml Bioruptor Microtubes New Cat No C30010011 Old Cat No WA 005 0500 is 100 ul When using lower volumes e g lt 50 ul 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 ul 10 ng ul recommended Using larger concentration e g 50 100 ng ul 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 qualty 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 inc
16. earing shearing recommended for DNA extraction Cat No C30010015 New Cat No C30010011 shearing Cat No C3001001 7 Old Cat No WA 005 0500 Cat No C30010014 15 ml Bioruptor Tubes amp sonication beads recommended for chromatin not recommended for DNA shearing Cat No C01020031 Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 17 Programmable DNA size distributions 1230 bp excellent reproducibility and high dsDNA al yields with Bioruptor Pico 1036 bp 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 848 bp Sample volume 100 ul Tubes 0 65 ml Bioruptor Microtubes New Cat No C30010011 Old Cat No WA 005 0500 Tube holder 0 65 ml tube holder for Bioruptor Pico Cat No B01200043 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 ul 10 ng ul 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 Sonication cycle am
17. ecommend to use the 0 1 ml tube holder amp tube adaptor Cat No B01200041 and the corresponding 0 1 ml Bioruptor Microtubes Cat No C30010015 0 1 ml tube holder amp tube adaptor for Bioruptor Pico Cat No B01200041 0 1 ml Bioruptor Microtubes Cat No C30010015 To use the tube holder remove the lower part by turning counterclockwise Then place microtubes into the unit Attach the lower part to the upper part of the adaptor lo guarantee homogeneity of 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 Standard operating conditions Sample volume 10 ul Tubes 0 1 ml Bioruptor Microtubes Cat No C30010015 Tube holder 0 1 ml tube holder amp tube adaptor for Bioruptor Pico Cat No B01200041 for 12 x 0 1 ml tubes Sonication buffer TE 10 mM Tris 1 mM EDTA pH 7 5 8 0 DNA concentration 10 ng ul recommended Samples are vortexed 10 15 sec and centrifuged 10 sec before shearing For optimal results samples should be stored on ice during 10 15 minutes prior to sonication Temperature 4 C Water cooler New Cat No B0201002 230V B02010003 115V B02010004 100V Old Cat No BioAcc Cool amp Single Cycle Valve for Water cooler Cat No B02020004 Sonication cycle and sonication time varies depending on desired DNA size see table Note
18. genode service center Improper or incorrectly performed maintenance or repairs will void the warranty Technical assistance amp ordering information Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA BEGE 5CIENCEIPARK 400 Morris Avenue Suite 101 Rue Bois Saint Jean 3 Denville NJ 07834 USA 4102 Seraing Ougr e Belgium Tel 1 862 209 4680 Jeu 324302205 Fax 1 862 209 4681 Fax 32 4 364 20 51 techsupport na diagenode com techsupport diagenode com orders na diagenode com orders diagenode com For a complete listing of Diagenode s international distributors visit www diagenode com en support distributors php For the rest of the world please contact Diagenode s a DIAGENODE BIORUPTOR PICO USER MANUAL Critical steps for maintenance and efficient Shearing 0 00 e eee ees 4 atPOGUGUG Ee 44224 otate es ete te set oe et eee eee teat eee eee a ee E 5 Bioruptor Pico technical specificationS ccc aanu eee ee eee eee hh 6 Getting to know your Bioruptor Pico system 000 c eect eee 7 EIoruptor Fico components OVEIVICW sur oo isn curacao Ug achs POR F RH REF RON d a Me eee i Borup eia ri oui ws ag ce aoe ag as aes ab heehee ee dae owas aes i Singe ale Valve Tor DIOP UBIO FIOO a esai pd aps Su ded OR DI ag e dct doo ge dde 8 SOM ANON uu METTI 8 DADOS IET aug eU nar Geld d bce Maia OO t E Sees aed Bg Ws ee OD Se 28 Bee Ian 7 WO TIONG Ss og eo sed 5s G Foe Be he es we ey i oe a ee
19. heared genomic DNA from separate experiments aiming at producing high quality DNA fragments compatible with lon 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 RF dx p 2000 ua p Panel B 13 cycles 30 sec ON 30 sec OFF cycles Expected fragment size 200 bp observed average fragment size is 202 2 bp CV96 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 3196 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 9696 mH acp x00 W pog poa 1335 fe All samples were analyzed on Bioanalyzer 2100 DNA High Sensitivity chips Ait o uU du Emu gm 17430 LE 10380 fal www diagenode com diagendtie PAGE 20 DIAGENODE BIORUPTOR PICO USER MANUAL 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 New Cat No C30010011 Old Cat No WA 005 0500 Pay attention not to damage the cap when closing the tubes since this could alter sonication results Sampl
20. ing 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 Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 9 Automatic temperature control The Water Cooler New Cat No B0201002 230V B02010003 115V B02010004 100V Old Cat No BioAcc CooU 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
21. l sonication accessories for Bioruptor Pico B01200016 Visit us at one of Diagenode s demo sites or discover our Automated Systems by performing some assays with the help of our R amp D and Technical Department www diagenode com DIAGENODE S A BELGIUM EUROPE LIEGE SCIENCE PARK DIAGENODE Rue Bois Saint Jean 3 4102 Seraina Belo HEADQUARTERS L5 55 Fax 32 4 364 20 51 orders diagenode com info diagenode com DIAGENODE INC USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 Tel 1 862 209 4680 Fax 1 862 209 4681 orders na diagenode com info na diagenode com FOR A COMPLETE LISTING OF DIAGENODE S INTERNATIONAL DISTRIBUTORS VISIT http www diagenode com company distributors php For rest of the world please contact Diagenode sa www diagenode com 2015 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors Bioruptor amp IP Star are registered trademarks of Diagenode Innovating Epigenetic Solutions MA BR PICO 1 2_09_15
22. ler 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 Water cooler Bioruptor Pico Single Cycle Valve 2 Place the Bioruptor on a bench Important Note Please make sure that the Bioruptor Pico is always placed on a level surface diagendide 3 Place the Water cooler below the Bioruptor Location Requirements The Water cooler must be located below the Bioruptor Pico minimum elevation difference 400 mm or 15 74 inch 4 Place the Single Cycle Valve on top of the Water cooler as shown in the image www diagenode com diagenctie PAGE 12 DIAGENODE BIORUPTOR PICO USER MANUAL 9 Insert the short red and blue tubing Into the outlets of the Water cooler and the lower red and blue nozzle connectors Es of the single cylce vavle box as shown in the image Respect the red and blue color codes 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 i 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 gener
23. ly random in AT or GC rich regions 2 The analytical process to determine fragment size has inherent variances for example gel electrophoresis and microfluidics based platform Therefore fragment distributions and peak values even from technical replicates may not appear identical If the sheared DNA sample will be resin or column purified or concentrated prior to analysis please remember to take out an aliquot for use as control prior to that step Column purification and concentration of the sheared DNA will generate a biased fragment distribution profile due to the inherent greater loss of the smaller DNA fragments 3 RNA contamination in genomic DNA preparation should be carefully removed using RNase DNase free enzymatic digestion since they might generate a biased fragment distribution profile on microfluidics based platform e g Agilent Bioanalyzer or alter sonication effiency www diagenode com diagendtie PAGE 26 DIAGENODE BIORUPTOR PICO USER MANUAL onsistent and highly reproducible chromatin shearing with the Bioruptor Pico 10 pl 300 pl HeLa cells are fixed with 1 molecular grade formaldehyde for 8 min at room temperature Nucleus isolation is performed using buffers and reagents of Diagenode s Chromatin Shearing Optimization kit Low SDS New Cat No C01020010 Old Cat No AA 001 0100 1x10e6 cells are then resuspended in 100 ul Shearing Buffer prior to chromatin shearing MW 10 15 20 500
24. ment 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 Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL Chromatin shearing from HeLa cells is performed according to Diagenode s Chromatin Shearing Optimization kit Low SDS protocol Cat No AA 001 0100 Nuclei are resuspended in buffer iS1 at a final concentration of 1x10e6 per 100 ul 2 ml nuclei aliquots are sonicated with the Bioruptor Pico in 15 ml Bioruptor Tubes Cat No C01020031 with or without sonication beads sonication beads are part of Cat No C01020031 for 10 20 or 30 cycles 30 sec ON 30 sec OFF Samples are vortexed every 10 cycle round beads beads MW 10 20 30 10 20 30 500 gt 100 2 MW corresponds to the 100 bp DNA molecular weight marker For shearing in a final volume of 2 ml the 15 ml Bioruptor Tubes are filled with sonication beads up to the third line of the graduation scale 0 3 ml corresponding to around 800 mg of beads Prior to use beads are washed by vortexing in 3 volumes of PBS All buffer is removed before adding the samples Following shearing samples are treated with RNase cocktail mixture at 37 C during 1 hour The sheared chromatin is then de crosslinked overnight and phenol chloroform purified as described in the kit manual 10 ul of DNA equivalent of
25. metal surface is fragile and requires a careful maintenance Use only soft sponge to remove traces Never use scratch scrub sponge since this would alter the ultrasonic wave emitter Surface 3 Water type Distilled water Supplementary Data Please note that there are three main sources of variation in both peak base pair size and distribution 1 The physical process of DNA fragmentation might not be entirely random in AT or GC rich regions 2 The analytical process to determine fragment size has inherent variances for example gel electrophoresis and microfluidics based platform Therefore fragment distributions and peak values even from technical replicates may not appear identical If the sheared DNA sample will be resin or column purified or concentrated prior to analysis please remember to take out an aliquot for use as control prior to that step Column purification and concentration of the sheared DNA will generate a biased fragment distribution profile due to the inherent greater loss of the smaller DNA fragments 3 RNA contamination in genomic DNA preparation should be carefully removed using RNase DNase free enzymatic digestion since they might generate a biased fragment distribution profile on microfluidics based platform e g Agilent Bioanalyzer or alter sonication effiency www diagenode com diagendtie PAGE 22 Innovating DIAGENODE BIORUPTOR PICO USER MANUAL NOTICE For DNA shearing we highly r
26. n broader size distribution or larger peaks Sample concentration Diagenode recommends using a DNA concentration of 10 ng ul Using larger or smaller concentrations may result in 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 10 15 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 leadingtoincorrect 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
27. novating Epigenetic Solutions 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 lon 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 Target Size 150 bp B Target Size 200 bp run FP 0 1329 J C Target Size 300 bp Fuj i P pu Jar 208 D Target Size 400 bp Tuy 3 i 5o BK am gb Ss amp Dd p Nx DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 19 Excellent reproducibility and programmable size distribution with Bioruptor Pico Panels A to D show size distributions of s
28. ntinuous 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 sample 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 Ultrasound disperse and break biomolecules The fragmentation of chromatin or Transducer DNA takes place as a consequence of this mechanical stress or shear Fig 1 Propagation in 0 65 ml tubes 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 www diagenode com diagendtie PAGE 6 DIAGENODE BIORUPTO
29. orrect 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
30. p 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 www diagenode com diagendtie PAGE 18 DIAGENODE BIORUPTOR PICO USER MANUAL Cycle condition On Off cycle time Cycle number 150 bp 30 30 200 bp 30 30 300 bp 30 90 400 bp 15 90 1000 bp D UV 30 13 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 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 E Lasgs7 B tarpe m auge 1 ET E E foren d 1 ra E i PAPAE je tee z 91 mI T mE ze 1 18 Iu Ladder gare eee pee J pepe d rape f mepi eee Donwvge Poel F pempe DLrsmpe 1 In
31. r Microtubes and 15 ml Cat No C01020031 Bioruptor Tubes amp 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 amp tube adaptor Cat No B01200041 0 65 ml tube holder Cat No B01200043 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 Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 5 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 co
32. ucibility with Bioruptor Pico using 0 1 ml Bioruptor Microtubes Panel A B and C show DNA size distributions of sheared human genomic DNA using standard operating conditions Panel A 200 bp after 20 cycles 30 sec ON OFF Average size 215 bp CV 6 696 Panel B 300 bp after 13 cycles 30 sec ON OFF Short centrifugation after 4 and 8 cycles added Average size 316 bp CV 4 596 Panel C 550 600 bp after 7 cycles 15 sec ON 90 sec OFF Short centrifugation after 3 cycles added Average size 576 bp CV 8 5 Panel A B and C peak electropherogram view PAGE 23 www diagenode com diagendtie PAGE 24 DIAGENODE BIORUPTOR PICO USER MANUAL 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 1 ml Bioruptor Microtubes Cat No C30010015 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 1 ml Bioruptor Microtubes Cat No C30010015 is 10 ul When using smaller volumes e g 10 ul not smaller than 5 ul 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 i
33. wer switch back side of sonication unit Innovating Epigenetic Solutions DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 15 CYCLE NUMBER TIME ON and TIME OFF are the Once the run started the RUNNING screen appears As parameters controlling sonication For changing these shown in Figure 4 the RUNNING screen indicates the parameters press the OK button First the TIME ON elapsed time ON time OFF and the number of cycles screen appears with the flashing number This parameter can be modified with the up T and down button Li 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 Figure 4 RUNNING Screen 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 the machine stopped during the second sonication cycle after Figure 1 Main Screen Te 30 sec of TIME ON and 8 sec of TIME OFF x l l LILI TIME TIME A ON OFF Cycle number reached CYCLE 4 cycles in this example START Bioruptor Pico will sonicate as shown Once parameters are set press START Figure 2 Figure 5 BREAK CYCLE Screen If the sonication process worked properly without any error messages the RUN OVER screen appears Figure
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