Bioruptor® Pico
Contents
1. 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 26 DIAGENODE BIORUPTOR PICO USER MANUAL Consistent and highly reproducible chromatin shearing with the Bioruptor Pico 10 pl 300 ul 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 gt 300 gt 5004 100 gt 1002 1002 A 10 pl volume B 100 ul volume C 300 ul 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 cy2. 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 e 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 sonication process e 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 ultrason
3. 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 FU A Programmable DNA size distribution and high reproducibility 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 35 100 150 200 300 400 500 600 1000 2000 10380 bp 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 B m 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 6 596 150 100 Panel A B and C peak electropherogram view 35 100 150 200 300 400 S00 600 1000 2000 10380 bp 300 400 500 600 1000 2000 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 24 DIAGENODE BIORUPTOR PICO USER MANUAL Important comments about DNA shearing The Diagenode ACT Adaptative Cavitation Transfer technol
4. diagenctie
5. diagenctie 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 diagenctie 3 Place the Water cooler below the Bioruptor Location Requirements he 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com 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 E 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 SW 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 co
6. 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 e ill 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 Tube holder Motorized lid Digital timer Distilled water e Transient overvoltage typically present on the MAINS supply e Degree of protection IP20 Installation overview Connector kit for Water Cooler diagenctle Single cycle valve Power cord n BIORUPTOR PICO diagenctie 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 11 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
7. AL Critical steps for emaintenance and efficient shearing 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 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 th
8. ME OFF oe E BBE BB TIME TIME 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 b BREAK CYCLE Screen 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 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 he 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 16 Em mum ii L u N IEEE m A m m MEE E deg cp j a Se DA EPA N pom t A py RS ers Orie les M mr LA E a 9 E Ue B UND web E i Ww u Q 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 s
9. NA 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 a
10. aia ain aa aa of sheared genomic DNA produced by varying the x 339 duration of sonication The different lanes depict a specific Bioruptor Pico run optimized to produce ME Siza n ED kan Ak n una SEE m a N specific mean sizes and size ranges for Next em 110 Generation DNA sequencing ene OOOO w Panel A peak electropherogram view Lanes 1 2 EM 150 bp Lanes 3 4 200 bp Lanes 5 6 300 bp Lanes GENE 7 8 400 bp EM Panel B gel virtual view 50 All samples were analyzed on Bioanalyzer 2100 ee nt nn EE ea a a ea using DNA High Sensitivity chip 30 30 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com A Target Size 150 bp FU 300 j 250 200 150 100 50 0 35 100 B Target Size 200 bp Lrul 250 200 150 100 35 100 150 C Target Size 300 bp FU 1504 100 50 35 100 150 200 D Target Size 400 bp FU 4 200 150 100 50 35 100 150 200 150 200 300 200 300 300 300 400 500 600 1000 2000 10380 bp j j 400 500 600 1000 2000 10380 bp po i j 400 500 600 1000 2000 10380 bp j J 400 500 600 1000 2000 10380 bp DIAGENODE BIORUPTOR PICO USER MANUAL Excellent reproducibility and programmable size distribution with Bioruptor Pico Panels A to D show s
11. alter sonication results Sample 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 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 D
12. cation 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com 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 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 diagenctie 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
13. cles 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 296 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 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 27 Consistent and highly reproducible chromatin shearing with the Bioruptor Pico 500 pl 2 ml Chromatin shearing from H
14. der 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 n 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 alte
15. diageng e Innovating Epigenetic Solutions Bioruptor Pico Sonication System Version 1 1 Bioruptor Pico Cat No B01060001 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 Imprope
16. e 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 Bioruptor 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 5 Introduction Diagenode s Bioruptor Pico uses a gentle method of sonication to retain the integrity of DNA and or biological complexes including chromati
17. eLa 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 500 000 cells are analyzed on a 2 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 settin
18. emperature 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 Recommended protocols are subject to change without notice Additional protocols are available on demand Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 23 Sonication table for small volume DNA Shearing Cycle conditions On Off time Cycle number 200 bp 307 30 20 cycles 250 bp 307 30 15 cycles 300 bp 90 730 13 cycles 550 600 bp 15 790 7 cycles 750 800 bp 15 790 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
19. entrifugation 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 A i l Ti Broad DNA size distribution excellent FU reproducibility and high dsDNA yields with Bioruptor Pico 250 Figure A shows different DNA size distributions m of sheared genomic DNA produced by varying T the duration of sonication from 2 separate experiments run to run variation The different e lanes depict 4 lanes from 2 different Bioruptor Pico runs optimized to produce specific mean 504 sizes corresponding to 150 200 300 and 400 bp based on the standard lon Torrent standard sample preparation size requirements B s s Figure B shows different DNA size distributions a xud
20. eral 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 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 e 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
21. gs 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com Description Bioruptor Pico sonication device for 0 65 ml tubes Bioruptor Pico sonication device for 1 5 ml tubes Single Cycle Valve for Bioruptor Pico Water cooler 0 1 ml tube holder amp tube adaptors for Bioruptor Pico 0 65 ml tube holder holder for Bioruptor Pico 1 5 ml tube holder for Bioruptor Pico 15 ml sonication accessories for Bioruptor Pico 0 1 ml Bioruptor Microtubes 0 65 ml Bioruptor Microtubes 1 5 ml Bioruptor Microtubes 15 ml Bioruptor Tubes 15 ml Bioruptor Tubes amp sonication beads Cat No B01060001 B01060002 B02020004 B02010002 230V B02010003 115V B02010004 100V Old BioAcc Cool B01200041 B01200043 B01200040 B01200016 C30010015 C30010011 C30010016 C30010017 C01020031
22. ic waves propagation resulting in Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com 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 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
23. 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 ul 1D or 30 pil 0 65 ml 100 ul 100 ul 1 5 ml 200 ul 300 ul 15 ml 900 ul 2ml Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com 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 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
24. is ws odour aoe owen Eee he Gee a eo hee ele nNnEEnEE HHHH Ae eed wee aera J TUO TO er a4 rae geen E e Gh daw bea ee ra e e E R oa E G EGQUIDMCREINStaUQtlON PT 10 Installation OVErVIEW 10 Installing the Bioruptor Pico system with Water Cooler WWW ka kk KK KK KK KK KK YR KK KK 11 Controlling the sonication e kaca ask Aa akla a ka Mak l ki kk DO dk kl Xel dan dan n W c a dl dk G 14 Tube holders and tub65 is ss aa a een ade ewra ka ew RU n xl wana a lead aa a w na da l lr warl a P aad 16 Standard protocol for DNA shearing kk kk kk kk kk kk kk kk KK kk kk kk a kk kk kok kk kk kk ka 17 Bieciuciiiprepeon euo Tr m Important comments about DNA shearing kk kk kk kk kk KK KK KK KK KK KK KK KRI KK KK YR 20 Small volume DNA shearing for Bioruptor Pico kk kk kk kk kk kK ees 22 te rale ap cola esas eue cae Pesce U bac ob ew oud SUGUEN EN Td TR qd EE NAN DM ides D ode 22 important comments about DNA Shearing s sesers s ks dina n oa el agencesbece kla cacee 20 Consistent and highly reproducible chromatin shearing with the Bioruptor Pico 20 Ordering InForrtidtiBEl es s pa dara a e diete ocu qoa bs n o dea le bab aca kok A d e en a da bi a dok heirs Back Cover Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 4 DIAGENODE BIORUPTOR PICO USER MANU
25. ize distributions of sheared 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 CV96 2 0796 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 4 796 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 96 All samples were analyzed on Bioanalyzer 2100 DNA High Sensitivity chips PAGE 19 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 20 DIAGENODE BIORUPTOR PICO USER MANUAL 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 New Cat No C30010011 Old Cat No WA 005 0500 Pay attention not to damage the cap when closing the tubes since this could
26. n 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 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 disperse and break biomolecules The fragmentation of chromatin or Transducer DNA takes place as a consequence of this mechanical stress or shear Ultrasound Fig 1 Propagation in 0 65 ml tubes With the Bioruptor Pico the entire volume of water present in the sonica
27. nnector 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 13 10 Press main switch on the front side of the Water cooler ES minichiller 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 ser and the arrow key wy of the control panel at the same time Display LED display status a E wire oe os Pump A Ww M Arrow keys SET key Start stop key zazay TEBER 12 Press Start Stop key to control the temperature Note See Water cooler manual for additional information Now you are ready to start Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 14 DIAGENODE BIORUPTOR PICO USER MANUAL Controlling the sonication D Digital Timer Allows the user to easily pr
28. nts overview Sonication bath Motorized lid Sonication unit Power cable EU Power cable US Tube holder Water Cooler To learn more please visit www diagenode com minichiller Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 8 DIAGENODE BIORUPTOR PICO USER MANUAL Single cycle valve for Bioruptor Pico CQ a 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 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 soni
29. ogram 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 power switch back side of sonication unit Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 15 Digital timer 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 Due 30 sec of TIME ON and 8 sec of TI
30. ogy process is highly reproducible However attention must be paid to the following treatment attributes to ensure best results e 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 e 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 in broader size distribution or larger peaks e Sample concentration Diagenode recommends using a DNA concentration of 10 ng ul Using larger or smaller concentrations may result in variable peak distribution e 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 e DNA Quality DNA quality and quantity must be considered carefully since bad quality and quantity DNA may have sev
31. paces with tubes containing the same volume of distilled water 0 1 ml tube holder amp tube adaptor for Bioruptor Pico Cat No B01200041 0 1 ml Bioruptor Microtubes recommended for chromatin amp DNA shearing Cat No C30010015 15 ml Bioruptor Tubes amp sonication beads recommended for chromatin not recommended for DNA shearing Cat No CO1020031 15 ml sonication accessories for Bioruptor Pico Cat No B01200016 1 5 ml tube holder for Bioruptor Pico Cat No B01200040 0 65 ml tube holder for Bioruptor Pico Cat No B01200043 eee 15 ml Bioruptor Tubes recommended for Tissue disruption for protein extraction Cat No C30010017 1 5 ml Bioruptor Microtubes recommended for chromatin not recommended for DNA shearing Cat No C30010016 0 65 ml Bioruptor Microtubes recommended for chromatin amp DNA shearing New Cat No C30010011 Old Cat No WA 005 0500 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 17 Standard protocols for DNA shearing Programmable DNA size distributions 1230 bp excellent reproducibility and high dsDNA 319 bp yields with Bioruptor Pico 1036 bp 234 bp Figure shows different DNA size distributions of sheared genomic DNA produced by varying
32. r 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 LIEGE SCIENCE PARK 400 Morris Avenue Suite 101 Rue Bois Saint Jean 3 Denville NJ 07834 USA 4102 Seraing Ougr e Belgium Tel 1 862 209 4680 lebe 4364 20 50 Fax trl ley 209 4691 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 PAGE 3 Contents Critical steps for maintenance and efficient Shearing cc kk kK KK KK ets 4 duigeeilei CT kode e Eee eee ot eee oe tues aaa e a tae e es 5 Bioruptor Pico technical specificationS kk kk kk kk kk Ek kk kK eee eee hr 6 Getting to know your Bioruptor Pico system 0 ccc eee kK kk kk kk eee kk ka 7 Bioruptor Pico components OVERVIEW uua aane C eed eda k ae ea 8 J RE KORE eee ERE ij Bloruptor Pico Water COO xau a x xa a xJ ne h RE RC ue E OCA RC a 9 qi WW dha a RR URL d A a Cw a 7 Single cycle valve Tor Bloruptor PICO uu poem dpsed dee I ded nte es c we CL OR e ww DR 8 seien c gn aided ese Sen Gaim ears E eee eto ete ae paid oe Ban Pee ede che Bete 8 MOTO ZED Wi
33. r sonication effiency Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 22 DIAGENODE BIORUPTOR PICO USER MANUAL Small volume DNA shearing for Bioruptor Pico NOTICE For DNA shearing we highly recommend 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 E s 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 To 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 T
34. 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 200 bp 150 200 500 600 i000 2000 10380 Standard operating conditions 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 amp 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 18 DIAGENODE BIORUPTOR PICO USER MANUAL Cycle condition Target size On Off cycle time Cycle number 150 bp 30 30 30 200 bp 30 30 13 300 bp 30 90 6 400 bp 15 70 7 8 1000 bp a p 7 8 For longer fragments 300 up to 1000 bp a short c
35. tion 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 6 DIAGENODE BIORUPTOR PICO USER MANUAL 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 Yes 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 Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 7 Getting to know your Bioruptor Pico system Bioruptor Pico compone
36. utomatic 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 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 e Water bath The sonication water bath is a critical component of the Bioruptor Pico sonication system Europe Diagenode sa LIEGE SCIENCE PARK Rue du Bois Saint Jean 3 4102 Seraing Ougr e Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR PICO USER MANUAL PAGE 21 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation resulting in broa
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