- Diagenode
Contents
1. DNA Qualtity DNA quality and quantity must be considered carefully since bad quality and quantity DNA may have several impacts on sonication and next gen sequencing downstream applications First DNA contamination e g from superfluous nucleic acids such as RNA small nucleic acid fragments excess proteins or other contaminating materials may interfere with DNA measurement method leading to incorrect DNA quantitation thus Also contaminating RNA in genomic DNA preparation might generate a biased fragment distribution profile on microfluidics based platform e g Agilent Bioanalyzer or alter sonication effiency Therefore it is highly recommended to use only high quality DNA when sonicating DNA for next gen sequencing library preparation The DNA sample to be processed should be highly pure having an 0D260 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 sonication bath During sonication especially when2. 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 Stop Button Ends sonication Intensity Setting Button Adjusts intensity of the i Intensity Level Meter ultrasonic waves generated Indicates the sonication Start Button intensity Begins sonication Power Switch Turns Bioruptor on and off CYCLE NUMBER TIME ON and TIME OFF and SONICATION INTENSITY are the parameters controlling the sonication First press or depending on the value to be modified The five flashing black squares move up or down Once you have selected the parameter to be modified press OK again The five flashing black squares disappear and 2 digits start flashing The digits can be changed by pressing or To select and save the correct number press OK to confirm or ESC to escape without saving the change IMPORTANT NOTE Minimum time of the OFF cycle 30 seconds Digital Timer After the introductory message the control screen shows the main sonication parameters in the first three lines CYCLE Number Time ON Time OFF and summary of actions in the last line see example below CYCLE Num 10 BIORUPTOR RUNNING aa Time ON 30sec RETA ae hese SE Bath 1 OFF Time OFF 30sec CYCLE Num 04 10 uir Press OK Modify TI PESE TIME TIME ON 4 OFF Cycle number reached p gt in thi l CYCLE 4 cycles
3. Version 1 1 Bioruptor Standard Bioruptor Standard for 1 5 ml tube holder B01010001 Bioruptor Standard for 1 5 amp 15 ml tube holder B01010002 Bioruptor Standard for 0 65 ml tube holder B01010003 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
4. 5 New Cat No B01200010 or 10 ml tubes holder Old Cat No UCD pack 10 New Cat No B01200012 with reflecting bar Sample volume 300 ul for 1 5 ml TPX microtubes 2 ml for 10 ml tubes Sonication buffer PBS with protease inhibitor cocktail Temperature Maintain at 4 C by using the Water Cooler Old Cat No BioAcc Cool New Cat No B02010002 230V B02010003 115V B02010004 100V or by using crushed ice Power setting H position High Sonication cycle 30 sec ON 30 sec OFF Total sonication time 10 min for Bioruptor Standard Note Please note that additional optimization might be required depending on the bacterial strain and growth phase Gram positive bacteria are more resistant to sonication than Gram negative bacteria because of the rigid cell wall Cells in log phase are less resistant than cells in stationary phase In order to preserve protein structure and activity avoid a long sonication Protocol 1 Collect cells by centrifugation at 1000 g for 10 min at 4 C 2 Wash twice with cold PBS 3 Resuspend cells in cold PBS to OD600 3 0 4 Transfer cell suspension to sonication tubes For optimal efficiency use the recommended sample volume 5 Sonicate at High Power for 10 min Bioruptor Standard 6 Centrifuge at 15 000 rpm for 15 min at 4 C 7 Separate the soluble fraction supernatant from the insoluble pellet 8 The pellet can be used for extraction of insoluble proteins with a denaturating buffer o
5. 5 0 65 ml tube holder Old Cat No UCD pack 0 5 New Cat No B01200010 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 Operating conditions Sample volume 100 ul Tubes 0 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc Tube holder 0 5 0 65 ml tube holder Old Cat No UCD pack 0 5 New Cat No B01200010 for 12 x 0 5 ml tubes Sonication buffer TE 10 mM Tris 1mM EDTA pH 7 5 8 0 DNA concentration 0 001 0 02 ug ul 0 01 ug ul recommended Europe Diagenode sa CHU Tour GIGA B34 39 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 17 Samples are vortexed 10 15 sec and centrifuged 10 sec before shearing For optimal results samples should be stored on ice during 10 15 min prior to sonication Temperature Maintain at 4 C by using ice chilled water and small amounts of crushed ice no more than 0 5 cm or use the Water Cooler Old Cat No BioAcc Cool New Cat No B02010002 230V B02010003 115V B020
6. Harvest cells by trypsinization Transfer cells in a tube containing 10 ml PBS RT and centrifuge 5 min at 1 300 rpm Keep the cell pellet and discard the supernatant Wash the cells again in PBS Note At this step cells might be counted 4 Add PBS to a final volume of 500 ul for a maximum of 10x10 6 cells for more cells perform the fixation in a separate tube Add formaldehyde to a final concentration of 196 mix gently and incubate for 8 10 min at RT with rotation Stop the cross linking reaction by adding glycine to a final concentration 0 125 M and incubate for 5 min at RT with 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 STANDARD USER MANUAL rotation 7 Wash cells 3 times with cold PBS 8 Resuspend cells in an appropriate volume of a Lysis buffer containing SDS 0 7 196 1x10 6 3x10 6 cells 300 ul are recommended for shearing in 1 5 ml tubes Lyse cells on ice for 5 10 min Vortex and centrifuge tubes before putting in Bioruptor Note Nuclei isolation is recommended when working with 3x10 6 cells to 10x10 6 cells Shearing ChIP kit from Diagenode is available for this purpose kch redmod 100 Diagenode 1 5 ml TPX microtubes are recommended for efficient chromatin shearing Old Cat No M 50050 or M 50001 New Cat No C30010010 Format 50 or 100 pc 9 Sonicate samples with Bioruptor Standard with refri
7. Sound pressure level dBA 79 dBA 100 80 ds 46 dBA diqgendtie 60 40 20 0 Bioruptor without Bioruptor with soundproof box soundproof box Soundproof Box Fig 4 Sound pressure of the Bioruptor without and with metallic soundproof box has been measured in a soundproof room Power Supply Adapter This unit provides the instrument with several signals used at the different stages during operation The power supply adapter is the heaviest part of the apparatus and needs particular handling caution The power supply adapter is switched on with the power button located on the back Power Supply Adapter Tube Holders Several sizes of tubes can be used with the Bioruptor Standard The minimum and maximum sample volume to be used with each container is given in the table below The 0 5 ml and 1 5 ml tubes can be simply closed and installed in the rotor For the sonication of larger volumes 10 ml 15 ml and 50 ml tubes a stopper with a metallic bar has to be used to reflect sound waves to produce a better resonance efficiency Tube Size Minimum Maximum 0 5 ml 50 ul 100 ul 1 5 ml 100 ul 300 ul 10 ml 500 ul 2ml 15 ml 500 ul 2ml Tube Holders 50 ml 3 ml 20 ml Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 10 DIAGENODE BIORUPTOR STANDARD USER MANUAL Equipment Installation The following pages contain information on ins
8. Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL resulting in broader size distribution or larger peaks Bath water should be pure distilled water changed regularly at least once per week 2 Sonication bath maintenance The sonication bath metal surface is fragile and requires a careful maintenance Use only soft sponge and distilled water to remove traces Never use scratch scrub sponge since this would alter the ultrasonic wave emitter surface Supplementary Data Please note that there are three main sources of variation in both peak base pair size and distribution 1 Sena 2 Sazan 3 The physical process of DNA fragmentation might not be entirely random in AT or GC rich regions 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 RNA c
9. 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 DNA takes place as a consequence of this mechanical stress or shear Ultrasound Transducer Fig 2 Propagation in 15 ml amp 50 ml tubes With the Bioruptor the entire volume of water present in the sonication bath is exposed to ultrasound allowing all the samples to be efficiently sonicated Figure 1 For larger tubes 15 ml or 50 ml the transfer of the ultrasound to the tubes is facilitated by a metallic bar in contact with the sample This metallic bar is not a probe no corrosion problems but reflects the ultrasound originated from the sonication bath and improves the sample sonication efficiency by a patented resonance system Figure 2 Noise level measurements amp precautions CEE noise data is not applicable to the Bioruptor s ultrasound emitter as no tests have been conducted on similar devices to date See listed noise levels as measured in the Accredited Acoustics Laboratory and precautions for the Bioruptor ba noise level in dBA 78 4 dB L dB Peak 87 6 dB m 1 Exposure limit value The exposure limit value is the maximum amount of noise an employee may be exposed to on any single day 8 hours Exposure beyond this limit presents a high ri
10. centrifuge tubes briefly before proceeding with the remaining time Make sure sonication bath is kept cool Once optimal conditions are reached use for all assays to assure reproducibility 1 5 ml per 15 ml tube 200 ul per 1 5 ml tube Check disrupted material on a 196 agarose gel 10 ul lane Run the gel slowly Gel electrophoresis of cross linked samples often gives smears on gel Also take several pictures of the gel to assure image quality The migration of large quantities of DNA on agarose gel can lead to poor quality pictures which do not reflect the real DNA fragmentation 1X TAE or TBE is preferred to 0 5X TAE which can lead to smears on gel Use a freshly prepared gel and fresh buffer Do not use a too big sample volume Rivero cross link from DNA after phenol chloroform extraction before loading on gel To obtain clearer image with accurate fragment size reversion of the cross linking is advised Do not load too much on a gel Do not load more than 5 ug lane Also treat the sample with RNAse Do not reuse an old gel Ordering information Bioruptor Models Bioruptor Standard for 1 5 ml tube holder Bioruptor Standard for 1 5 amp 15 ml tube holder Bioruptor Standard for 0 65 ml tube holder Cooling System Water Cooler including continuous valve for Bioruptor Consumables 1 5 ml TPX Microtubes 1 5 ml TPX Microtubes 15 ml TPX Microtubes 0
11. in this example START The display shows cycle 4 of 10 Bioruptor NGS will sonicate as shown Buttons and their functions Button A Pause restart after pausing Button B Press and hold this button during sonication to display T1 total on time per cycle and T2 total off time per cycle ESC Cancels previous selection OK Validate selection Decrease selected parameter move down Increase selected parameter move up Once all parameters are selected and confirmed press START Once the run is started BIORUPTOR RUNNING is displayed on the control screen Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 14 DIAGENODE BIORUPTOR STANDARD USER MANUAL Overheat Shutdown The ultrasound source is sensitive to high temperatures which means that increases in temperature diminish the sonication efficiency and can damage the device The production of ultrasound generates heat which Is transferred into the sonication bath To avoid excessive overheating of the ultrasound source the Bioruptor Standard Cat No contains a temperature sensor We recommend that the water in the sonication bath remains between 4 C 10 C 39 F 50 F for optimum sonication Note he sensor does not measure the water temperature inside the ultrasound bath where the samples are submerged Once a critical limit is reached a warning is displayed on the control screen Depending on t
12. 10004 100V Power setting L position Low 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 Cycle conditions On Off times in sec Number of cycles 1250 bp 15 90 2 cycles 950 bp 15 90 4 cycles 750 bp 30 90 3 cycles 550 bp 30 90 5 cycles 400 bp 30 90 6 cycles 350 bp 30 90 8 cycles 300 bp 30 90 10 cycles 250 bp 30 90 15 cycles 200 bp 30 90 30 cycles 150 bp 30 30 70 cycles recommended to use a lab timer to set time precisely 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 18 DIAGENODE BIORUPTOR STANDARD USER MANUAL Important comments about DNA shearing The Diagenode ACT Adaptative Cavitation Transfer technol
13. 5 ml Bioruptor Microtubes for DNA Shearing DIAGENODE HEADQUARTES Diagenode sa BELGIUM EUROPE Tel 32 4 364 20 50 Fax 07 4 304 20 51 orders diagenode com info diagenode com diagengte Innovating Epigenetic Solutions Tube Holders 0 5 0 65 ml tube holder for B01010001 Bioruptor Standard Bioruptor Plus B01010002 1 5 ml tube holder for Bioruptor Standard amp Bioruptor Plus B01010003 10 ml tube holder for Bioruptor Standard amp Bioruptor Plus 15 ml tube holder for Bioruptor Old Cat No BioAcc Cool Standard amp Bioruptor Plus New Cat No B02010002 230V B02010003 115V B02010004 100V 50 ml tube holder for Bioruptor Standard amp Bioruptor Plus Old Cat No M 50050 or M 50001 New Cat No C30010010 Format 50 or 100 pc Old Cat No M 50050 or M 50001 New Cat No C30010010 Format 50 or 100 pc Old Cat No M UN 15 New Cat No 30010010 Format 50 pc or 1000 pc Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc Diagenode Inc USA NORTH AMERICA Tel 1 862 209 4680 Fax 1 862 209 4681 orders na diagenode com info na diagenode com Old Cat No UCD pack 0 5 New Cat No B01200010 Old Cat No UCD pack 1 5 New Cat No B01200010 Old Cat No UCD pack 10 New Cat No B01200012 Old Cat No UCD pack 15 New Cat No B01200013 Old Cat No UCD pack 50 New Cat No B01200014 For a complete listing of Diagenode s int
14. Diagenode s products are performed by our approved Diagenode 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 Avenue de l h pital 1 400 Morris Avenue Suite 101 Tour GIGA 3rd Floor Denville NJ 07834 USA 4000 Liege Belgium Tel 1 862 209 4680 Tel 32 4 364 20 50 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 http www diagenode com en support distributors php For the rest of the world please contact Diagenode s a DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 3 Contents Critical Steps for Maintenance and Efficient Shearing kk kk KK KK KK K RR ee 4 nalan ee ee i ee ee ee ee ee ee ee ee eee 5 Bioruptor Technical Specifications 0 c cc eee eens 6 Getting to Know Your Bioruptor System kS kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk ka 7 Bioruptor Components Overview kk kk kk kk kk kk kk kK KK KK ss 7 SONIC AION Old gt gt gt gt YC lt bLe gt aaaa beh ce oe amp one r r awh ehh e ee wees S J MOUGEIZEG WIG af aee a a EE EN 6 oe ee Rea Ges eae oe A Hee ee eee we oe eke oe Rees 8 Metalic Sgt TOQ DON c c sas pai etre De RR h kd na Weha Adee G
15. ced in the tube Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL Introduction Diagenode s Bioruptor Standard 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 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 produces better and more consistent results than with harsher sonication methods Up to Ultrasound 12 closed tubes can be sonicated in parallel and the continuous rotation Transducer of tubes allows even distribution of the energy for efficient sonication The Bioruptor enables automation of sonication guaranteeing higher reproducibility of results Fig 1 Propagation in 0 5 ml tubes and 1 5 ml tubes The effect of ultrasound on biological samples The Bioruptor sonication system uses ultrasound to create focused mechanical stress to lyse cells or shear DNA or chromatin Ultrasound waves pass through the sample expanding and contracting the liquid During expansion negative pressures pull the molecules away from one
16. doing long sonication runs the temperature must also be controlled Note The permanent installation of the Bioruptor in a cold room is possible although not sufficient to avoid the temperature increase due to sonication This location would only replace the pre cooling step described above Automatic temperature control A recirculating water cooler is used to guarantee the automatic temperature control of the sonication bath during the whole sonication process This water cooler Old Cat No BioAcc Cool New Cat No B02010002 230V B02010003 115V B02010004 100V produces a regular water flow with a constant water level in the tank Sonication time Minor adjustments in cycle number may be made to optimize results for various sample types and concentrations The table above listing the cycle parameters and numbers 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 Sonication bath The sonication sonication bath is a critical component of the Bioruptor 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation Europe Diagenode sa CHU
17. e control of the sonication bath during the entire sonication process Figure 3 The water flow shouldn t exceed 500 ml min to allow optimal resonance The Water Cooler features two pumps and produces a regular water flow to maintain a constant water level in the tank e minichiller Sonication Bath Control unit Water Cooler Fig 3 Setup of the Bioruptor Standard in combination with the Water Cooler Note The water cooler must be located below the Bioruptor Note You may permanently install the Bioruptor in a cold room though this is not sufficient to avoid the temperature increase during sonication The cold room would only eliminate the need for the precooling step described above Motorized Lid The motorized lid along with the blue or white 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 blue or white gear plate Avoid the immersion of the motor into the water Do not heat the blue or white plastic as it will warp Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 432 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 9 Metallic Soundproof Box This metallic soundproof box absorbs more than 30 dBA generated by the ultrasonic sonication bath Figure 4
18. e size accuracy What do smears indicate How much DNA should load and is RNAse treatment necessary What should my running buffer concentration be Will using an old gel cause problems the fixation cells 1 ml m 10e6 10x TE le 300 ul What is the key buffer component How long is the shearing What is the earan cycle 30 seconde ON 30 OFF What is the best volume tube for shearing What kind of gel should Answers 1 Correct formaldehyde concentration in fixation is critical Fix for 10 min with a time course when needed It is possible to fix for as little as 5 min depending on your protein of interest for subsequent ChIP assays teense at room temperature Fixation can be performed at 4 C RT and 37 C Make sure you perform the fixation step at the right temperature Weso ihe Hed celie propa the fixed cells properly Make sure you get rid of ALL the formaldehyde Use glycine to stop Do not use too many cells in the cell lysis buffer Lyse about 5x 10e6 3x 106 30x 106 cells 1 ml Include detergent in buffer The HighCell ChIP kit is compatible with cell numbers up to 10 million cells in small volumes Do not use a too high cell concentration Quality and quantity of detergent is important Perform a time course for chromatin shearing It is possible to shear from 5 30 min If 30 interrupt sonication after every 10 min and
19. ent it is necessary to start the sonication process with cold water and to keep it at 4 C during the sonication process Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 8 DIAGENODE BIORUPTOR STANDARD USER MANUAL Manual temperature control e A precooling of the sonication bath tank 15 min before starting the first round of sonication is advised This prevents the water from heating too quickly due to thermal inertia i e when the tank and the ultrasound generating elements are stored at room temperature To precool simply add crushed ice and then fill with cold water up to the indicated level red line on the water level sticker e Every 10 min replace crushed ice The ice floating in the water should not exceed 0 5 cm and the total water level water amp ice should be exactly at the indicated water level Fill entirely a 250 ml beaker with crushed ice Pour ice carefully into your sonication bath which is already filled with to the red fill line Remove approximately 130 ml of water without ice Carefully adjust water level to the indicated mark red line by removing or adding water using a pipette Automatic temperature control The Water Cooler Old Cat No BioAcc Cool New Cat No B02010002 230V B02010003 115V B02010004 100V can be used in combination with the Standard Connector Kit continuous valve to guarantee the automatic temperatur
20. ernational distributors visit http www diagenode com en support distributors php For the rest of the world please contact Diagenode s a 2010 Diagenode Inc All rights reserved Bioruptor is a registered trademark of Diagenode The content of this document cannot be reproduced without prior permission of the authors Bioruptor and IP Star are registered trademarks of Diagenode Inc MESSRS 25 065 1s
21. f choice Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 22 DIAGENODE BIORUPTOR STANDARD USER MANUAL Efficient cell disruption with Bioruptor Cell suspensions were sonicated for different periods of time ranging from 5 to 20 min Two types of tubes were tested Diagenode s 1 5 mL TPX tubes Old Cat No M 50050 or M 50001 New Cat No C30010010 Format 50 or 100 pc and 10 ml tubes Old Cat No AS 100 New Cat No C30010012 Format 100 pc or 500 pc The efficiency of cell disruption was initially determined by measuring optical density at 600 nm The results indicated that the number of intact cells decreases rapidly with increasing sonication time After only 5 min of sonication a significant number of cells were disrupted Fig 1 Similar results were observed using the Live Dead BacLight kit data not shown which allows the quantification of live cells with intact membranes and discrimination from cells with damaged membranes Thus efficient cell disruption is observed after 5 10 min of sonication Cell disruption post sonication 120 100 80 60 40 20 Hi 10 ml tube 1 5 ml TPX tube OD600 fold changes 0 min 5 min 10 min 20 min Figure 1 Effect of sonication on cell disruption The number of intact cells after sonication was determined by measuring optical density at 600 nm Optical density of the cell culture before sonication 0 min is arbit
22. gerated sonication bath or crashed ice sonication bath for 10 20 30 cycles of 30 sec ON and 30 sec OFF at HIGH setting Briefly vortex and centrifuge tubes after each run of 10 cycles 10 Centrifuge samples at 14000 rpm for 5 min at 4 C and transfer the supernatant into a new tube Use an aliquot of sheared chromatin equivalent of 100 000 500 000 cells for analysis of shearing perform a reversal of cross links and analyze on agarose gel The remaining chromatin might be kept at 80 C Shearing of chromatin from suspension cell lines Note Cells growing in suspension culture are known to be difficult to shear Nuclei extraction is recommended before sonication Do not use very dense cell suspension for sonication 1 Cross link chromatin with 1 fresh formaldehyde for 8 10 min at RT 2 Stop the cross linking reaction by adding glycine to the final concentration 0 125 M for 5 min at RT with gentle rotation 3 Wash cells 3 times with cold PBS 4 Extract cell nuclei and use isolated nuclei for shearing Shearing ChIP kit from Diagenode is available for this purpose kch redmod 100 5 Resuspend nuclei in an appropriate volume of Lysis buffer containing SDS 196 1x10 6 3x10 6 cells 300 ul are recommended for shearing in 1 5 ml tubes Lyse nuclei on ice for 5 10 min Vortex and spin down tubes before putting in Bioruptor Note Diagenode 1 5 ml TPX microtubes are recommended for efficient chromatin shearing Old Cat No M 50050
23. he situation different screens pop up as described below 1 If START is pressed and the instrument stops after a couple of seconds showing the following screen blinking the instrument could not start a new run due to the ultrasound source s temperature Causes e Instrument has been used several times in a row recently without breaks e Instrument is stored in a place exposed to direct sun e Room temperature is too hot What to do e Store the instrument in another place if the current one does not meet the installation specific requirements e Allow for longer breaks between uses e Place crushed ice in sonication bath to help the cool ultrasound source Machine too hot to start a new run See Manual B ESC 26096 2 During arun if the instrument stops and the following screen pops up the critical temperature has been reached To protect the ultrasound source from damage the instrument has stopped and is standing by to be restarted after the ultrasound source has cooled Causes e Instrument has been running too long without breaks RISK OF DAMAGE Machine stoped at What to do Cycls XX XX See Manual e Store the instrument in another place if the current one does not meet the installation specific requirements e Allow for longer breaks between uses e Place crushed ice in sonication bath to help to cool down the ultrasound source B ESC 9 amp 3 If the warning message a
24. ient Shearing General warnings N e DO NOT turn on the instrument without water e DO NOT tilt the sonication bath To change the water use either the plastic pump or a beaker Water level sonication bath e The sonication bath must be filled with distilled water only to the fill line do not use deionized water Fill line replacement stickers can be obtained by contacting Diagenode Change water at least once per week Water temperature sonication bath e Optimal water temperature for sonication is 4 C Sample temperature should not exceed 10 C e Methods to maintain the temperature ce add small amounts of crushed ice no more than 0 5 cm to the bath every 10 min Water Cooler Old Cat No BioAcc Cool New Cat No B02010002 230V B02010003 115V B02010004 100V the flow rate of the circulating water cooler cannot exceed 500 ml per min for optimal sonication Magnetic Ultrasound Emitter Maintenance e The ultrasound waves are created from a series of magnets that are attached to the sonication bath This system is very sensitive to the heat generated during a run e o not exceed 1 hour of total sonication per run It is critical that the machine is allowed to cool at least 20 min 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 m
25. il orders na diagenode com PAGE 12 DIAGENODE BIORUPTOR STANDARD USER MANUAL Installing the Bioruptor Standard 1 Open the boxes and unpack all components 2 Place sonication bath in 3 Remove the rubber cap upper left hole marked machine cable from the back of the front of the soundproof soundproof box and feed the control unit cable through the hole The fitting will secure box the cable to the sound proof box Turn the front section counterclockwise to seal Be sure there is enough slack in the cable 4 Plug the control unit cable 5 Place the motor lid on the top of the sonication bath and 6 Place the sonication bath into into the sonication bath connect it Make sure that the cable of the motor lid is the soundproof box connected as shown in the image triangles have to match 7 Fill the sonication bath up to the red fill line with 8 Connect the power supply adapter to the control unit distilled water only Do not use deionized water with the power cable Place the control unit on top of the soundproof box Plug the power cable into the outlet and switch on the power switch on the back of the control unit Now you are ready to start Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 432 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 13 Controlling the Sonication
26. l info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 7 Getting to Know Your Bioruptor Standard Bioruptor Components Overview Control Unit Sonication bath Motorized lid Soundproof Box Power Cable Control Unit Cable Tube holder example Power Supply Adaptor 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 bath must remain upright at all times especially when moved Tilting the sonication bath 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 tank 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 Use only distilled water to fill the tank Do not use deionized water 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 generate heat Drop off in efficiency will occur above 10 C To ensure preservation of the samples and to prevent damage to the instrum
27. ning tube hard plastic polyethylene ref Corning 430304 can be used as well as soft plastic Polypropylene ref Corning 430290 but you should stick to one kind as transfer of ultrasonic waves is different for the different tube types hard plastic is more efficient Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 16 DIAGENODE BIORUPTOR STANDARD USER MANUAL Holding Plates included with any tube holder O 0 rings for 10ml 15ml 50ml tube holders The holding plate for 10 ml and 1o ml tubes can accommodate up to 6 tubes For 50 ml tubes the sample holding plate can accommodate up to 3 tubes The holding plates should always be completely filled to guarantee homogeneity of shearing By removing the black knob it is possible to replace the O ring The complete tube holder chip including O ring can be sterilized in the autoclave After more than 20 autoclave sterilizations O the O ring might need to be replaced visit www diagenode com Standard protocols DNA shearing For DNA shearing we highly recommend to use the tube holder for 0 5 0 65 ml tubes Old Cat No UCD pack 0 5 New Cat No B01200010 and the corresponding Bioruptor 0 5 ml Microtubes for DNA Shearing Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc 0 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc 0
28. oa ac kk SUR PRO IR RU Xe 8 Alk d n he RC 6az D 9 Power Supply Adapter s ssie madra ed eao k lala eh 7 DX RTT y EGUIDMICNE MNS erisia rarena rir EAE nE EAE EAE REEE TE EEEE EEPE RENS 10 lastallation OVEINIOW ss caedem REP AERKREOEESREES RSIUBe REG ANA RM E R la Bara dank Q 2 a dn y SU aes 10 Installing the Bioruptor Standard eiie kK kK KK KK KK KK KK KK KK KK KK KK KK KK KK Ka 12 Controlling the Sonication saos eoo areis kk kk kk kk c kk KK KK KK KK kk kk kk kk kk lk kk kk kk lk lk kk kk kk lk kk 13 Tube Holders amp TUDES supe ha wn 0 pi a edid rd cie de pe di asa dake k di don n SIE Sd d n d ds dl pad 15 Standard pi Olt0CO0LlS3 scii uy sans ELSE lk l kb band r all aran he K toes ki dene d kn i le dr de od d da dil a and ERE 16 DINE aa e o_rnremBU gt N _ IU BF7HI7IEU7H 7N NM_ gt I gt gt gt 2 X2XKX KXMAKxRMK x2KxKx2x2xxKxKxKxx Ep gt gt I zzMAMDZZJMMJJMmMMN T 16 tv Wez yy Q J N o EN TE LT LETT TII DO DOTT 19 Doc erie Gell bS UD yan ln kla pace e K d r y ee dran w a qi dle a Kn Grab A ED 0 21 TroupleS hooting TTG 23 Ordering Informati n ss 246 ace on iran eR kra kay Wa dr Ee E ERE RR RR EERDER K wa 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 STANDARD USER MANUAL Critical Steps for Maintenance and Effic
29. ogy 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 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc 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 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc is 100 ul When using lower volumes e g 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 a 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 10 15 min before sonication has also been shown to improve reproducibility
30. ontamination 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 Chromatin shearing Critical points for chromatin shearing Chromatin shearing efficiency varies on cell type Each cell type might need additional protocol optimization The extent of cross linking is critical for the efficient disruption of fixed cells and also affects DNA yield and average size of chromatin fragments Over cross linked chromatin will not produce small fragments even by prolonged sonication Fix cells for 8 10 min at RT always stop the reaction by glycine and wash 2 3 times with ice cold PBS Cell density affects the sonication efficiency Do not use too dense cell suspension Optimal density is about 1 3x10 6 100 ul of sonication buffer SDS is a key component of sonication buffer for chromatin shearing Include 0 7 1 of SDS in your sonication buffer Fresh methanol free formaldehyde for fixation Shearing of chromatin from adherent cell lines For the adherent cells we recommend to first harvest cells by trypsinization and perform chromatin cross linking In a cell suspension rather than on dishes as it results in a better reproducibility and consistency between experiments 1 2 Discard medium to remove dead cells and wash cells by adding cold PBS
31. or M 50001 New Cat No C30010010 Format 50 or 1000 pc 6 Sonicate samples with Bioruptor Standard with refrigerated sonication bath or crashed ice sonication bath for 10 20 30 cycles of 30 sec ON and 30 sec OFF at HIGH setting Briefly vortex and spin down tubes after each run of 10 cycles 7 Centrifuge samples at 14000 rpm for 5 min at 4 C and transfer the supernatant into a new tube Centrifuge samples at 14000 rpm for 5 min at 4 C and transfer the supernatant into a new tube Use an aliquot of sheared chromatin equivalent of 100 000 500 000 cells for analysis of shearing perform a reversal of cross links and analyze in agarose gel The remaining chromatin can be kept at 80 C Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 21 Bacterial Cell Disruption For cell lysis we highly recommend using 1 5 ml TPX microtubes Cat No M 50050 or 10 ml tubes Old Cat No AS 100 New Cat No C30010012 Format 100 pc or 500 pc and the corresponding tube holders Old Cat No UCD pack 1 5 New Cat No B01200010 and UCD pack10 To guarantee homogeneity of sonication the tube holders should always be completely filled with tubes Operating conditions Tubes 1 5 ml TPX microtubes or 10 ml tubes Tube holder 1 5 ml tube holder Old Cat No UCD pack 1
32. oving sonication bath Validated tubes for the Bioruptor Standard e DNA shearing 0 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc e Chromatin Shearing 1 5 mL TPX microtubes Old Cat No M 50050 or M 50001 New Cat No C30010010 Format 50 or 100 pc and 15 ml TPX tubes Old Cat No M UN 15 New Cat No 30010009 Format 50 pc Other tubes might be used but will require additional optimization Once a brand of tube is optimized switching brands may result in changes in sonication efficiency Fitting 0 5 ml or 1 5 ml tubes in the corresponding tube holder 1 Place the tubes on the corresponding tube holder 0 5 0 65 ml tube holder Old Cat No UCD pack 0 5 New Cat No B01200010 or 1 5 ml tube holder Old Cat No UCD pack 1 5 New Cat No B01200011 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 the lid 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 Fitting of 15 or 50 ml tubes in the corresponding tube holder 1 Loosen both the blue and the black top prior to placing the metallic reflecting bar in the tube 2 First tighten the blue ring then the black top This will ensure the O ring is properly pla
33. ppears at the bottom of the BIORUPTOR RUNNING screen see below it means that the critical temperature is about to be reached Causes e Instrument has been running too long without breaks BIORUPTOR RUNNING zw wz 1 r r kr kr What to do CYCLE Num xx xx e Reduce the number of consecutive cycles or split protocols into two runs separated by WARNING gets hot SE NL CN RR OK Keep cold water in the sonication bath while running to help keep the ultrasound e Q gt source at low temperature Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 15 Tube holders amp tubes Tube holders Available for 0 5 0 65 ml 1 5 ml 10 ml 15 ml and 50 ml To use adaptor tube unit remove the lower part of the microtube holder by turning counterclockwise Then place microtubes in the unit Attach the lower part to the upper part of the adaptor To guarantee homogeneity of chromatin shearing the tube holders should always be completely filled with tubes To ensure reproducibility always use the same brand of tubes e The 2 ml polypropylene tubes thin walled should not be used with the Bioruptor 1 5 ml tubes in TPX plastic that provide better ultrasound transfer rates and more efficient sonication are available from Diagenode see price list These tubes should be
34. rarily set to 100 Sheared DNA is released during bacterial sonication The disruption of bacterial cells by sonication releases DNA with maximum recovery after only 5 min of treatment Fig 2 AJ The released DNA is fragmented with fragment size dependent on sonication time Fig 2 B A dsDNA release 40 35 34 6 34 2 33 6 30 25 20 15 10 concentration pg ml 5 min 10 min 15 min 20 min B M m n hmin 10min 15min 20 min I Figure 2 Effect of sonication on DNA release Figure A The DNA concentration in each sample after sonication was quantified with the DNA BR assay kit Invitrogen Figure B An aliquot of each sample before 0 min and after sonication was run in a 1 596 agarose gel stained with SybrSafe and visualized in UV light Lane M represents a 100 bp ladder Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 432 4 364 20 50 Mail info diagenode com Troubleshooting Bioruptor Chromatin Shearing FAQs Critical Steps Questions What is the formaldehyde final concentration How long is the fixation step Wiatisihe tempers te is the temperature to use for fixation Aroihewadlesaler the washes after fixation important How can l achieve complete cell disruption What is the amount of cells per shearing trial to use What is the best temperature for shearing use to determin
35. sk to the user L 8h 87 dB A XPOSURE m P 200 Pa 140 dB C referring to 20 Pal PEAK PAGE 5 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 6 DIAGENODE BIORUPTOR STANDARD USER MANUAL 2 Upper exposure Action value The exposure action value is the upper daily limit of noise exposure Exposure beyond this value requires employers to take action to limit user exposure n 8h 85 dB A EXPOSURE 140 Pa 137 dB C referring to 20 Pa PEAK 3 Lower exposure action value r 8h 80 dB A EXPOSURE U 112 Pa 135 dB C referring to 20 Pa PEAK Use of Bioruptor 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 engths for a long period of time Bioruptor Technical Specifications Bioruptor 115 V 4 2A US 230V 2 1A EU 50 60 HZ Available for 0 5 1 5 10 15 amp 50 ml tubes 0 5 ml tubes 12 1 5 ml tubes 6 l tubes 6 l l Number of samples to be processed simultaneously 10m lam 50 ml tubes 3 tubes 6 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mai
36. talling your Bioruptor Standard 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 back side of the control unit and secure the unit against being re energized No special tools are required Three 3 square meters are needed to set up the Bioruptor Devices are designed to be safe under the following conditions e Indoor use e Altitude up to 2 000 meters e Operating external temperature 0 C to 25 C e Maximum relative humidity 80 e Transient overvoltage typically present on the MAINS supply e Degree of protection IP20 Installation overview 0 5 0 65 amp 1 5 ml jera tube adaptor Flexible format 10 ml 15 m TIR tube adaptor 50 ml Emm tube adaptor Motorized lid Rotation e Metallic soundproof box Start button Flexible Level meter control Digital timer Power plug must be grounded POLLUTION DEGREE 2 Normally only non conductive pollution occurs However occasionally a temporary conductivity caused by condensation is expected Never install this equipment in a place where environmental conditions and warnings mentioned above are infringed Flexible format Fits into current workflow with standard tubes Scales with flexible sample volume Rotation Prevents contamination wi
37. th closed tubes Continuous rotation through sonication bath guarantees equal distribution of energy Flexible control Easy to program Power range effectively disrupts samples Gentle ultrasound Saupe Gentle ultrasound method e Cooling diagenc cte minichiller Power cord preserves sample Power button Sonication bath Q Cooling Output selector switch Gentle ultrasound Cooling system maintains Stop button integrity of sensitive samples Fig 5 Schematic installation overview of the Bioruptor Standard in combination with the Water Cooler Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 11 Power Supply Adapter The power supply adapter must be plugged into the power grid An IEC lead is provided IMPORTANT WARNING Ensure that your power inlet behind the power supply adapter shows the right voltage corresponding to your area Otherwise switch it using a narrow blade screwdriver When connecting cables always be sure pins are Keep dot facing up Once plugged in secure properly aligned Note the indexing pins on the control by turning 90 clockwise unit cables for precise mating alignment until a click is heard Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Ma
38. used for chromatin shearing not DNA shearing e Any 0 5 ml or 1 5ml tube can be used for chromatin sheraring although shearing efficiency is increased by using the hard plastic tubes TPX hard plastic available from Diagenode See catalog numbers on price list e For DNA shearing only 0 5 ml Bioruptor Microtubes Old Cat No WA 004 0500 New Cat No C30010013 Format 500 pc can be used e The complete adaptor including O ring can be sterilized in the autoclave After more than 20 autoclave sterilizations the O ring might need to be replaced see price list for spare parts 0 5 0 65 ml tube holder 1 5 ml tube holder 10 ml tube holder Old Cat No UCD pack 0 5 Old Cat No UCD pack 1 5 Old Cat No UCD pack 10 New Cat No B01200010 New Cat No B0120001 1 New Cat No B01200012 15 ml tube holder Old Cat No UCD pack 15 New Cat No B01200013 The tube holders for 15 ml tubes are available for Falcon amp Corning tubes If you use another brand of tubes use the one which fits in the holder the best When using the 15 ml tubes do not forget to insert the aluminium ring to ensure an optimal position of the tube during sonication 50 ml tube holder Old Cat No UCD pack 50 New Cat No B01200014 The tube holders for 50 ml tubes are available for Falcon tubes blue and for Corning tubes orange If you use another brand of tubes use the one which fits in the holder the best Note The quality of the 50 ml Cor
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