- Diagenode
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1. STANDARD USER MANUAL PAGE 19 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 1 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 Cat No M 50050 or M 5001 6 Sonicate samples with Bioruptor Standard with refrigerated water bath or crashed ice water 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 chr2. Version 1 1 Bioruptor Standard Cat No UCD 200 Guarantee Limited one year global warranty Diagenode guarantees all products from any manufacturing defects as we rigorously test all products to meet strict quality standards Diagenode warrants that all standard components of its instruments will be free of defects in materials and workmanship for a period of one 1 year from the date that the warranty period begins unless the original quotation or accompanying documentation states a different warranty period All warranty periods begin on the date of delivery and apply only to the first purchaser of the product If a manufacturing defect arises and a valid claim Is received within the warranty period Diagenode at Its discretion will repair or replace the product in accordance with the warranty terms and conditions stated herein In case of repair or replacement of a product under warranty Diagenode will cover the expenses to return the repaired or replacement product This warranty covers only manufacturing defects and does not cover any damage caused by misuse lack of compliance to recommendations stated in the manual neglect accidents abrasion or exposure to extreme temperatures chemical solvents or acids We strongly recommend that maintenance or repairs of Diagenode s products are performed by our approved Diagenode service center Improper or incorrectly performed maintenance or repairs will void the warranty Techn
3. unmethylated DNA control TSH2B GAPDH mc green 003 hMeDIP kit Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 hmC Ab fast qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected 1 pg 2 or 3 days 1 5 h Hydroxymethylated methylated and unmethylated BAC clones Hydroxymethylated DNA control methylated DNA control unmethylated DNA control Sfil for genomic DNA 16 AF 104 0016 AF 110 0016 AF 111 0016 MagBisulfite kit Gives precise information on methylation status of single cytosines High conversion rate 499 DNA purification based on magnetic beads and compatible with SX 8G IP Star Automated System qPCR sequencing microarray Ing 1 ug 3 5 hours 2h Bisulfite specific primer pair 24 AF 106 0024 diagengde Innovating Epigenetic Solutions Ordering information Bioruptor Models Connector Kits UCD 200 TM 1 5 ml Single Cycle Valve for Bioruptor Water Cooler E VB 101 0001 Bioruptor Standard UCD 200 TO 1 5 ml amp 15 ml Bioruptor Plus amp NGS UCD 200 TS 0 5 0 65 mi Dual Cycle Valve for Bioruptor Water Cooler VB 100 0001 UCD 300 TM 1 5 ml Bioruptor Twin Bioruptor Plus UCD 300 TO 1 5 ml amp 15 ml Tube Holders UCD 300 TS 0 5 0 65 ml 0 5 0 65 ml tube holder for Bioruptor Standard jen i i pack 0 Bio
4. 10 ml 500 ul 2 ml 15 ml 900 ul 2 ml 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 installing 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 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 A La conductivity caused by condensation is expected e Maximum relative humidity 80 e Never install this equipment in a place where environmental conditions and warnings mentioned above are infringed e Transient overvoltage typically present on the MAINS supply e Degree of protection IP20 Installation overview 0 5 0 65ml amp 1 5 ml Ep tube holder 10 ml 15 ml tube holder NE 50 ml tube holder he Bioruptor Water Co
5. Automated DNA methylation kits Auto MethylCap kit 1 Allows to capture fractions of methylated DNA by CpG density Includes control primer pairs for assessment of capture efficiency qPCR linear amplification and genome wide analysis such as microarray and sequencing 2 TSH2B GAPDH 48 AF Auto01 0048 Auto MeDIP kit 1 Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 meC Ab fast qPCR linear amplification and genome wide analysis such as microarray and sequencing 2 Methylated and unmethylated BAC clones Methylated DNA control unmethylated DNA control 16 or 100 AF Auto0 AF Auto0 AF Auto0 AF Auto0 1 A016 1 G016 1 A100 1 G100 1 Validated on SX 8G IP Star and SX 8G IP Star Compact Automated Systems 2 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit Auto hMeDIP kit 1 Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 hmC Ab fast qPCR linear amplification and genome wide analysis such as microarray and sequencing 2 Hydroxymethylated methylated and unmethylated BAC clones Hydroxymethylated DNA control methylated DNA control unmethylated DNA control Sfi1 for genomic DNA 16 AF Auto02 0016 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sa
6. BIORUPTOR STANDARD USER MANUAL Controlling the Sonication INTERVAL ON OFF f Intensity level e meter _ Kul JIR a Multi timer D Be Intensity setting button Total timer CYCLE NUMBER TIME ON TIME OFF and sonication INTENSITY are the key parameters controlling the sonication Multi timer The control unit allows the automatic production of ON OFF cycles to preserve the samples from rapid heating due to the ultrasound energy This is achieved by the Multi timer located on the upper right part of the control unit INTERVAL ON OFF INTERVAL ON OFF INTERVAL ON OFF PERMANENT 30 sec ON 30 sec 30 sec ON 90 sec 15 sec ON 180 sec OFF cycle OFF cycle OFF cycle The setting of the OFF time is done by the green needle which can be handled by turning the external surface of the dial The setting of the ON time is done by the red needle which can be handled by turning the internal knob of the dial The default unit is the minute The time units min or sec can be modified by using a screw driver on the screws directly above the ON and OFF Three examples of ON and OFF cycles set on the multi timer are shown above Total analog timer The Bioruptor Standard is activated by the clockwise rotation of this dial It has 4 positions 5 min 10 min 15 min permanent for any total sonication time higher than 15 min T
7. Optimal water temperature for sonication is 4 C Sample temperature should not exceed 10 C e Methods to maintain the temperature Ice add small amounts of crushed ice no more than 0 5 cm to the bath every 10 minutes Bioruptor Water Cooler Cat No BioAcc Cool the flow rate of the circulating water cooler cannot exceed 500 ml per minute for optimal sonication Magnetic Ultrasound Emitter Maintenance e The ultrasound waves are created from a series of magnets that are attached to the water tank This system is very sensitive to the heat generated during a run e Do not exceed 1 hour of total sonication per run It is critical that the machine is allowed to cool at least 20 minutes between runs Damage resulting from non compliance to manual Instructions will void the warranty and shorten the lifespan of the machine e Ultrasound Emitters can be damaged by tilting or jarring the machine Exercise care If moving water tank Validated tubes for the Bioruptor Standard e DNA shearing Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 e Chromatin Shearing 1 5 ml TPX microtubes Cat No M 50050 or M 50001 and 15 ml TPX tubes Cat No M UN 15 Others 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 co
8. below Handling The water bath must remain upright at all times especially when moved Tilting the water 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 Distilled water should be used to fill the tank Replacement stickers can be obtained from Diagenode Water temperature The water in the water 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 instrument 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 Bioruptor s tank 15 minutes 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
9. fragment distribution profile on microfluidics based platform eg 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 eg Proteinase K digested 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 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 leg 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 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
10. protocol with agarose beads optimized for transcription factors Transcription factors and co factors Yes 1 million 3 days 2h Cell lysis chromatin shearing IP anti H3 K4me3 BMX c fos beta actin myoglobin exon 2 Not included 18 kch redTBP 012 Histone ChIP kit Standard protocol with agarose beads optimized for histones and their modifications Histones and histone modifications Yes 100 000 3 days 2h Cell lysis chromatin shearing IP anti TBP or RNA Pol Il 2 GAPDH 0 5 kb idem 1 kb c fos beta actin myoglobin exon 2 Not included 18 kch orgHIS 012 1 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit 2 Please mention your choice of antibody on your purchase order OneDay ChIP kit Protocol using agarose beads quick and ready to use on large quantities of sheared chromatin fast All DNA protein interactions Yes 1 5 2x 10e6 2h IP DNA purification anti IgG rabbit DNA purifying slurry 60 180 kch oneDIP 060 kch oneDIP 180 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 26 DIAGENODE BIORUPTOR STANDARD USER MANUAL Features Downstream applications Amount of DNA rxn Total Time of Assay Handling time Internal controls Control PCR primer pairs rxns per kit Cat No
11. sample before 0 min and after sonication was run in a 1 5 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 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR STANDARD USER MANUAL Troubleshooting Bioruptor Chromatin Shearing FAQs EWTE HET E HEE EEE ana Fixation Cell lysis Number of cells shearing buffer volume Shearing buffer Shearing step Checking for high quality shearing on an agarose gel What is the formaldehyde final concentration How long is the fixation step What is the temperature to use for fixation Are the washes after fixation important How can achieve complete cell disruption What is the amount of cells per shearing trial to use What is the key buffer component How long is the shearing What is the optimal cycle What is the best temperature for shearing What is the best volume tube for shearing What kind of gel should use to determine 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 1 Fix for 10 minutes with a time course when needed Fix at room temperature Wash the fixed cells properly Make sure yo
12. 004 0500 is 100 ul When using lower volumes leg 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 e Sample concentration Diagenode recommends using a DNA concentration ranging between 1 and 20 ng pl 10 ng pl recommended Using larger concentration eg 50 100 ng ul may result in broader peaks or 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 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 leg from superfluous nucleic acids such as RNA small nucleicacid fragments excess proteins or other contaminating materials may interfere with DNA measurement method leading to incorrect DNA quantitation thus Also contaminating RNA n genomic DNA preparation might generate a biased
13. 11 Controlling the S NICA ION vasc ed bee eh orion ada pdas da 12 Using UDC HOSTS s si sk k o ot eee aras ita e ida d j a es ba dia ita A dek ar ds d d hed 13 Standard Protocole aa kasa xan kal de hall a acar ama PURER aba a AE S poe eee And a 15 DRAN a dute pde sh ste linda dota ds wie le Gpe ls does eae Sees 15 Chromatin SST nespia eun pene per ao qe Es po idea pde AO us Bae caer 16 Bacterial Gell DIS UDS orar Pd PG oe Rae woe ee wea es 20 Iroupe ROOM xr xuda sheen aca ba de as j dk ava d ar di deda nea be d e akar ne d wa akar vee ae ane 23 Related PIT OCT S as sue roo kla and d b a la a lr oh di A ak daran dir dh ar d jk Ar Q di io aso oie 24 Ordering MOT IT 3U ON ace Ana su sis nak k heed dkm ak d l la a eee tones 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 Efficient Shearing General warnings IN e DO NOT turn on the instrument without water e DO NOT tilt the water bath To change the water use either the plastic pump or a beaker Water level water bath e The water bath must be filled with distilled water to the fill line Fill line replacement stickers can be obtained by contacting Diagenode Change water at least once per week Water temperature water bath e
14. UARTES Diagenode sa BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA Tel 32 4 364 20 50 Tel 1 862 209 4680 Fax 32 4 364 20 51 Fax 1 862 209 4681 orders diagenode com orders na diagenode com info diagenode com info na 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 2010 Diagenode Inc All rights reserved Bioruptor is a registered trademark of Diagenode MA BR STANDARD_30_03_12 The content of this document cannot be reproduced without prior permission of the authors Bioruptor and IP Star are registered trademarks of Diagenode Inc
15. 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 rotation Wash cells 3 times with cold PBS Resuspend cells in an appropriate volume of a Lysis buffer containing SDS 0 7 1 1x10 6 3x10 6 cells 300 ul are recommended for shearing in 1 5 ml tubes Lyse cells on ice for 9 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 Cat No M 50050 or M 50001 7 Sonicate samples with Bioruptor Standard with refrigerated water bath or crashed ice water 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 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
16. ccessory keeps the sample tubes in constant rotation and ensures optimal position in the water bath during sonication When in motion do not hamper the rotation of the blue gear plate Avoid the immersion of the motor into the water Do not heat the blue 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 32 4 364 20 50 Mail info diagenode com Metallic Soundproof Box DIAGENODE BIORUPTOR STANDARD USER MANUAL PAGE 9 This metallic soundproof box absorbs more than 30 dBA generated by the ultrasonic waterbath Figure 4 q G _ diagenctie Tube Holders Sound pressure level dBA 79 dBA 100 80 46 dBA 60 40 20 0 Bioruptor without Bioruptor with soundproof box soundproof box Fig 4 Sound pressure of the Bioruptor without and with metallic soundproof box has been measured in a soundproof room 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 Holders Tube Size Minimum Maximum 0 5 ml 50 ul 100 pl 1 5 ml 100 ul 300 pl
17. d characterized in house For a complete listing of Diagenode s antibodies please visit www diagenode com for more information 4 Chromatin Immunoprecipitation kits Automated ChIP kits Auto ChIP kit 1 Auto Histone ChIP seq kit 1 Optimized for working with histone antibodies in ChIP seg experiments saving time maximum reproducibility All DNA protein interaction saving Features time maximum reproducibility Optimized for All DNA protein interactions Histones and histone modifications Downstream applications qPCR qPCR sequencing arrays 2 Amount of cells IP 1 000 1 million 1 000 10 million Total Time of Assay 1 day 1 day Handling time 30 min 30 min Buffers and reagents IP DNA Isolation IP Control antibodies anti IgG rabbit anti IgG rabbit DNA purification DNA isolation buffer DIB rxns per kit 16 or 100 16 or 100 AB Auto01 A016 AB Auto02 AU16 AB Auto01 G016 AB Auto02 G016 Cat No AB Auto01 A100 AB Auto02 A100 AB Auto01 G100 1 Validated on SX 8G IP Star and SX 8G IP Star Compact Automated Systems 2 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit SX 86 IP Star Compact Automated System AB Auto02 G100 Auto Transcription ChIP kit 1 Optimized for working with TF anbosies saving time maximum reproducibility Transcription factors and co factors qPCR sequencing arrays 2 1 000 10 mill
18. described above Automatic temperature control A recirculating water cooler is used to guarantee the automatic temperature control of the water bath during the whole sonication process This water cooler cat No BioAcc cool produces a regular water flow with a constant water level in the tank 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 Water bath The sonication water bath is a critical component of the Bioruptor sonication system 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation resulting in broader size distribution or larger peaks Bath water should be pure distilled water changed regularly 2 Water bath maintenance The water 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 Suppl
19. ementary 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 3 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 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 eg Agilent Bioanalyzer or alter sonication effiency PAGE 17 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 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 li
20. ery 10 minutes and centrifuge tubes briefly before proceeding with the remaining time Make sure waterbath Is kept cool Once optimal conditions are reached use for all assays to assure reproducibility Do not use a too big sample volume Reverse 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 24 DIAGENODE BIORUPTOR STANDARD USER MANUAL Related Products Diagenode develops and sells premium products for Epigenetics research that provide industry leading sensitivity and consistency Diagenode offers a number of kits for Chromatin Immunoprecipitation ChIP and DNA methylation assays like Methylated DNA immunoprecipitation MeDIP MethylCap MBD and Bisulfite conversion MagBisulfite kit The Bioruptor with its powerful yet gentle ultrasound technology allows for consistent shearing a narrow size range of sheared DNA or chromatin and sample preservation necessary for successful experiments Antibodies Diagenode offers a large selection of optimized ChIP amp ChIP seg grade as well as MeDIP amp MeDIP seq grade antbodies that we have developed an
21. he maximum time is 15 minutes The time set will equal the sum of the ON and OFF cycles Alternatively turning the timer knob counterclockwise will set the Bioruptor Standard on a permanent position always cycles ON and OFF To stop this process manually set the timer knob on to the vertical position Intensity setting button 3 power settings are available for the sonication L Low M Medium and H High Intensity level meter allows visualization of the power delivered by the Control Unit to the water bath relative scale 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 Tube holders amp tubes Tube holders Available for 0 5 0 65 ml 1 5 ml 10 ml 15 ml and 50 ml tubes To use the 0 5 0 65 ml amp 1 5 ml tube holder remove the lower part of the holder by turning counterclockwise Then place tubes into the unit Attach the lower part to the upper part of the tube holder To guarantee homogeneity of sonication the tube holders should always be completely filled with tubes To ensure reproducibility always use the same brand of tubes Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 are recommended for DNA shearing e 1 5 ml TPX microtubes Cat No M 50050 M 50001 provide better ultrasound transfer rates and more eff
22. ical Assistance amp Ordering Information Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA Avenue de Uhopital 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 techsupportOdiagenode com orders naUWdiagenode com ordersOdiagenode 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 4 kk kK KK KK KK KRK ees 4 nalan lt ecos tasas ocaso testeo taste r He 5 Bioruptor Technical Specifications 0 c cc eee eens 6 Getting to Know Your Bioruptor System kk kk kk eee kk kk kk kk kk kk a 7 Bioruptor Components Overview 7 VEEN eorna aa a e e ee HE L KOO 0 90 RRA Ln nn E e eee E e E E E E NS 8 Metalll SOMIMG DP TOQ ION caras ked n Ea de as a A aie he Wn gt 9 JT eno lo a e si ia on eed wi og Bete A e e A E wee eee A a Be ee ee Bee Sees 9 EQUIPpmentinstatla llo si s 4k sc dk xuma ayn ALEA dl da caba and X ei a i 10 nata al lon Overview serca rra pro HOES Sheard k WIR BA xl Av le AAA A KES 10 Stalling the Bioruptor SISTE 2 ececsepra mi d K k al h AI RA EE AR
23. icient sonication These tubes should be used for chromatin shearing not DNA shearing Any 0 5 ml or 1 5ml tubes can be used although shearing efficiency might be lower The 2 ml polypropylene tubes thin walled should not be used with the Bioruptor e The complete tube holder including O ring can be sterilized in the autoclave After more than 20 autoclave sterilizations the O ring might need to be replaced visit www diagenode com for more information 0 5 0 65 ml tube holder 1 5 ml tube holder 10 ml tube holder Cat No UCD pack 0 5 Cat No UCD pack 1 5 Cat No UCD pack 10 15 ml tube holder Cat No UCD pack 15 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 Cat No UCD pack 50 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 Corning 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
24. igid 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 UCD200 300 or 15 min Bioruptor XL 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 of choice 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 Efficient cell disruption with Bioruptor Cell suspensions were sonicated for different periods of time ranging from 5 to 20 minutes Two types of tubes were tested Diagenode s 1 5 ml TPX tubes M 50001 and Diagenode s 10 ml tubes AS 100 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 minutes of sonication a significant number of ce
25. ion 1 day 30 min IP anti IgG rabbit 16 or 100 AB Auto03 A016 AB Auto03 G016 AB Auto03 A100 AB Auto03 6100 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 Manual ChIP kits PAGE 25 Features Optimized for Suitable for ChlP seq and ChIP on chip Amount of cells IP Total Time of Assay Handling time Buffers and reagents for Control antibodies Control PCR primer pairs DNA purification rxns per kit Cat No LowCell ChIP kit Magnetic bead based protocol for all DNA protein interaction fast increased DNA yield All DNA protein interactions Yes 1 1 000 1 million Cell lysis chromatin shearing IP DNA purification anti IgG rabbit human TSH2B c fos myoglobin exon 2 DNA isolation buffer 16 or 48 kch maglow A16 kch maglow G16 kch maglow A48 kch maglow G48 HighCell ChIP kit Magnetic bead based protocol Ideal to recover large amount of DNA transcription factors ChIP on chip and to avoid biais due to amplification steps All DNA protein interactions Yes 1 1 10 million Cell lysis chromatin shearing IP DNA purification anti IgG human TSH2B GAPDH promoter DNA isolation buffer 16 kch mahigh A16 kch mahigh G16 Transcription ChIP kit Standard
26. lls 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 minutes of sonication Cell disruption post sonication 120 100 M 10 ml tube 1 5 ml TPX tube 80 60 40 20 0D600 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 arbitrarily set to 100 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 Sheared DNA is released during bacterial sonication The disruption of bacterial cells by sonication releases DNA with maximum recovery after only b minutes of treatment Fig 2 A The released DNA is fragmented with fragment size dependent on sonication time Fig 2 B dsDNA release 40 35 30 25 20 15 10 concentration pg ml 5 min 10 min 15 min 20 min M 0 min 5b m n 10min 15min 20min 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
27. more efficient PAGE 13 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 Holding Plates included with any tube holder O O rings for 10ml 15ml 50ml tube holders The holding plate for 10 ml and 15 ml tubes can accommodate up to 6 tubes For o0 ml tubes the sample holding plate can accommodate up to 3 tubes The holding plates should always be completely f lled 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 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 Standard protocols DNA shearing For DNA shearing we highly recommend to use the tube holder for 0 5 0 65 ml tubes Cat No UCD pack 0 5 and the corresponding Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 6 0 5 0 65 ml tube holder Cat TE No UCD pack 0 5 M To use the tube holder remove the lower part by tu
28. n 1250 bp 15 90 3 9 min 950 bp 15 90 7 min 750 bp 30 90 6 min 550 bp 30 90 10 min 400 bp 30 90 12 min 350 bp 30 90 16 min 300 bp 30 90 20 min 250 bp 30 90 30 min 200 bp 30 90 60 min 150 bp 30 30 70 min 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 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 e Tubes At present the recommended tube vessels are the Diagenode s Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 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 Diagenode s Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA
29. nking 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 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 L 2 J Discard medium to remove dead cells and wash cells by adding cold PBS Harvest cells by trypsinization Transfer cells in a tube containing 10 ml PBS RT and centrifuge 5 minutes 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 1 mix gently and Incubate for 8 10 min
30. oler Sample tube holding plate Motor lid Soundproof box Multi timer Intensity level meter Intensity setting button Power cord Water bath Power cord Fig 5 Schematic installation overview of the Bioruptor Standard System in combination with the Bioruptor Water Cooler Control unit Total timer 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 2 Place water bath in front 3 Remove the rubber cap upper left hole marked machine cable from the back of the of the soundproof box soundproof box and feed the control unit cable through the hole 4 Plug the control unit 5 Place the motor lid on the top of the water bath and 6 Place the water bath nto the cable nto the water bath connect t soundproof box 7 Fill the water bath up to the red fill line with d stilled 8 Plug the control unit 9 Plug power cable into water cable into the control the control unit unit 11 Plug the power cable into the outlet and switch on the power switch on the back of the control unit 10 Place the control unit on the top of the soundproof box 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 12 DIAGENODE
31. omatin 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 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 Bacterial Cell Disruption For cell lysis we highly recommend using 1 5 ml TPX microtubes Cat No M 50050 or 10 ml tubes Cat No AS 100 and the corresponding tube holders Cat No UCD pack 1 5 and UCD pack10 To guarantee homogeneity of sonica tion 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 Cat No UCD pack 1 5 or 10 ml tubes holder Cat No UCD pack 10 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 Bioruptor Water Cooler Cat No BioAcc Cool or by using crushed ice Power setting H position High Sonication cycle 30 sec ON 30 sec OFF Total sonication time 10 min for UCD200 300 15 min for Bioruptor XL 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 r
32. onicators 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 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 waterbath 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
33. rning 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 Bioruptor 0 5 ml Microtubes for DNA Shearing Cat No WA 004 0500 Tube holder 0 5 0 65 ml tube holder Cat No UCD pack 0 5 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 Samples are vortexed 10 15 sec and centrifuged 10 sec before shearing For optimal results samples should be stored on ice during 10 15 minutes prior to sonication Temperature Maintain at 4 C by using ice chilled water and small amounts of crushed ice no more than 0 5 cm or use the Bioruptor Water Cooler Cat No BioAcc Cooll 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 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 Cycle conditions On Off times in sec Totas P un j mi
34. rresponding tube holder 0 5 0 65 ml tube holder Cat No UCD pack 0 5 or 1 5 ml tube holder Cat No UCD pack1 5 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 placed 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 PAGE 5 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 water bath to generate indirect sonication waves which emanate from an ultrasound element below the water tank Because the system is gentler than other s
35. rt Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com Manual DNA methylation kits Features Suitable for Amount of DNA rxn Total Time of Assay Handling time Internal controls Control PCR primer pairs rxns per kit Cat No MethylCap kit Allows to capture fractions of methylated DNA by CpG density magnetic beads permit fast and sensitive capture Includes control primer pairs for assessment of capture efficiency qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected lg 1 day 2h TSH2B GAPDH 48 AF 100 0048 MagMeDIP kit Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 meC Ab fast qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected lg 2 or 3 days Lon Methylated and unmethylated BAC clones Methylated DNA control unmethylated DNA control TSH2B GAPDH 10 or 48 mc magme A10 mc magme 048 MeDIP kit Quality control using internal controls high specificity monoclonal 5 meC Ab agarose beads fast qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected 1 pg 2 or 3 days 2h Methylated and unmethylated BAC clones Methylated DNA control
36. ruptor Twin UCD 400 TM 1 5 ml Bioruptor Plus amp Bioruptor NGS i UCD 400 TO 1 5 ml amp 15 ml 1 5 ml tube holder for Bioruptor Standard amp UCD k15 pack 1 Bioruptor XL UCD 500 TM 1 5 ml Bioruptor Plus UCD 500 TO 1 5 ml amp 15 ml 10 ml tube holder for Bioruptor Standard amp b Bioruptor NGS UCD 600 TS 0 5 0 65 ml Siocunter Pius UCD pack 1 Cooling System 15 ml tube holder for Bioruptor Standard amp PI UCD pack 15 Bioruptor Water Cooler including continuous valve Bioruptor Plus BioAcc cool for Bioruptor 50 ml tube holder for Bioruptor Standard amp PI UCD pack 50 Peristaltic pump including standard connectors TWI pump Bioruptor Plus ert Se 0 5 0 65 ml tube holder for Bioruptor Twin TWI pack 0 5 r l 1 Seal TEC Micretubes M 50001 1 5 ml tube holder for Bioruptor Twin TWl pack 1 5 E 15 pal TEW Milerotubas M 50050 10 ml tube holder for Bioruptor Twin TWl pack 10 PENR j 15 mi TPX Microtubes M UN 15 15 ml tube holder for Bioruptor Twin TWl pack 15 ae j Bioruptor 0 5 ml Microtubes for DNA Shearing WA 004 0500 spelen O Ao A Pesce a j Bioruptor NGS 0 65 ml Microtubes for DNA Shearing WA 005 0500 gt 0 69 ml tube holder for Bioruptor XL A Ju 1 5 ml tube holder for Bioruptor XL XL pack 1 5 10 ml tube holder for Bioruptor XL XL pack 10 15 ml tube holder for Bioruptor XL XL pack 15 50 ml tube holder for Bioruptor XL XL pack 50 DIAGENODE HEADQ
37. sample This metallic bar is not a probe no corrosion problems but reflects the ultrasound originated from the waterbath 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 E nolse 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 risk to the user L 8h 87 dB A XPOSURE m P 200 Pa 140 dB C referring to 20 uPa PEAK 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 L 8h 85 dB A EXPOSURE P 140 Pa 137 dB C referring to 20 Pa PEAK 3 Lower exposure action value L 8h 80 dB A EXPOSURE u 112 Pa 135 dB C referring to 20 Pa PEAK Use of Bioruptor by pregnant
38. 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 minutes replace crushed Ice The ice floating in the water should not exceed 0 5 cm and the total water level water ice should be exactly at the indicated water level Fill entirely a 250 ml beaker with crushed ice Pour ice carefully into your sonication water bath which is already filled with water 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 Bioruptor Water Cooler Cat No BioAcc Cool guarantees the automatic temperature control of the water bath during the entire sonication process Figure 3 The cooling system features two pumps and produces a regular water flow to maintain a constant water level in the tank tT VS TT ST DAA Bioruptor Water Cooler Water bath Control Unit Fig 3 Setup of the Bioruptor Standard System in combination with the Bioruptor Water Cooler 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 gear plate a
39. u get rid of ALL the formaldehyde Use glycine to stop the fixation Do not use too many cells in the cell lysis buffer Lyse about 5x 10e6 cells 1 ml 1x 10e6 10x 10e cells 300 pl 3x 106 30x 106 cells 1 ml Include detergent in buffer Perform a time course for chromatin shearing 30 seconds ON 30 seconds OFF AR 1 5 ml per 15 ml tube 200 ul per 1 5 ml tube Check disrupted material on a 1 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 PAGE 23 Correct formaldehyde concentration in fixation is critical lt is possible to fix for as little as 5 minutes depending on your protein of interest for subsequent ChIP assays Fixation can be performed at 4 C RT and 37 C Make sure you perform the fixation step at the right temperature 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 It is possible to shear from 5 30 minutes If 30 interrupt sonication after ev
40. 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 Bioruptor Technical Specifications Power Supply 115 V 4 2A US 230V 2 1A EU 50 60Hz Control unit dimensions 150 W x 285 D x 195 H mm Sonication unit dimensions water bath 175 W x 160 D x 265 H mm Soundproof box dimensions 350 W x 350 D x 500 H mm Water bath volume 750 ml Timer Analog Total weight 30 kg Tube holder Available for 0 5 1 5 10 15 amp 50 ml tubes 0 5 ml tubes 12 1 5 ml tubes 6 Number of samples to be processed simultaneously 10 ml tubes 6 15 ml tubes 6 50 ml tubes 3 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 7 Getting to Know Your Bioruptor Standard System Bioruptor Components Overview Control Unit Water bath Motorized lid Soundproof Box Control Unit Cable Tube holder example Water bath The water bath is a critical component of the instrument The generators below the tank produce ultrasonic waves which are then transferred through water The water bath requires special handling and care as described
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