Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl...

Europe - Diagenode s.a. / orders@diagenode.com / info@diagenode.com // North America - Diagenode Inc. / orders.na@diagenode.com / info.na@diagenode.com

www.diagenode.com

Standard protocols DNA shearing for Bioruptor® Pico

Standard operating conditions

Sample volume: 100µl

Tubes: 0.65mlBioruptor®Microtubes(Cat.No.WA-005-0500)

Tube holder: 0.65mltubeholderforBioruptor®Pico(Cat.No.B01200042)for12x0.65mltubes

Sonication buffer: TE(10mMTris,1mMEDTA,pH7.5-8.0)

DNA concentration:1-20ng/µl(10ng/µlrecommended)

Samplesarevortexed(5-10sec)andcentrifuged(10sec)beforeshearing.For optimal results samples should be stored on ice during 5-10 minutes prior to sonication.

Temperature: 4°C – Water Cooler (Cat. No. BioAcc-Cool) & Single Cycle Valve (Cat. No.B02020004)

Sonication cycle & total sonication time:variesdependingondesiredDNAsize(seetable)

Note: Recommended protocols are subject to change without notice. Additional protocols areavailableondemand.

125 bp

135 bp

183 bp

200 bp

234 bp

319 bp

476 bp

696 bp

848 bp

1036 bp

1230 bp

Programmable DNA size distributions, excellent reproducibility, and high dsDNA yields with Bioruptor® Pico

Figure shows different DNA sizedistributions of sheared genomicDNAproducedbyvaryingthedurationof sonication. The different curvesdepictaspecificBioruptor®PicoNext-Generationrun,optimizedtoproducespecific mean sizes and size rangesforNext-Generationsequencing.All samples were analyzed onBioanalyzer 2100 using DNA HighSensitivitychip.

www.diagenode.com

Target sizeCycle condition

(On/Off cycle time)Cycle number

150bp 30’’/30’’ 30

200bp 30’’/30’’ 13

300bp* 30’’/90’’ 6

400bp* 15’’/90’’ 7-8

1000bp* 5’’/90’’ 7-8

*Forlongerfragments(300upto1000bp),ashortcentrifugationstepafterhalfofthecyclenumberscansignificantlyimprovetheresults.Protocolsforothersizeranges(incl.longerfragmentsupto1300bp)areavailableonrequest.

Broad DNA size distribution, excellent reproducibility, and high dsDNA yields with Bioruptor® Pico

Figure A shows different DNA sizedistributions of sheared genomic DNAproduced by varying the duration ofsonicationfrom2separateexperiments(run-to-runvariation).Thedifferentlanesdepict4lanesfrom2differentBioruptor®

Picoruns,optimizedtoproducespecificmean sizes corresponding to 150, 200,300 and 400 bp (based on the standardIonTorrentstandardsamplepreparationsizerequirements).

Figure B shows different DNA sizedistributions of sheared genomic DNAproduced by varying the duration ofsonication. The different lanes depict aspecific Bioruptor® Pico run, optimizedto produce specific mean sizes andsize ranges for Next-Generation DNAsequencing.

Panel A: peak electropherogram view.Lanes 1,2: 150 bp; Lanes 3,4: 200 bp;Lanes5,6:300bp;Lanes7,8:400bp.

PanelB:gelvirtualview

AllsampleswereanalyzedonBioanalyzer2100usingDNAHighSensitivitychip.

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.

www.diagenode.com

A.TargetSize:150bp

C.TargetSize:300bp

B.TargetSize:200bp

D.TargetSize:400bp

Excellent reproducibility and programmable size distribution with Bioruptor® Pico

PanelsAtoDshowsizedistributionsofsheared genomic DNA from separateexperimentsaimingatproducinghigh-qualityDNAfragmentscompatiblewithIonTorrentstandardsamplepreparationprotocols(150,200,300and400bp).

Panel A: 30 cycles, 30 sec ON/30 secOFFcycles.Expectedfragmentsize:150bp, observed average fragment size is150.6bp(CV%:2.07%).

Panel B: 13 cycles, 30 sec ON/30 secOFFcycles.Expectedfragmentsize:200bp, observed average fragment size is202.2bp(CV%:4.7%).

PanelC:6cycles,15secON/90secOFFcycles.Expectedfragmentsize:300bp,observedaveragefragmentsizeis291.3bp(CV%:4.31%).

PanelD:7cycles,15secON/90secOFFcycles.Expectedfragmentsize:400bp,observedaveragefragmentsizeis406.9bp(CV%:7.96%).

All samples were analyzed onBioanalyzer 2100 DNA High Sensitivitychips.

www.diagenode.com

Important comments about DNA shearing

The Diagenode ACT (Adaptative Cavitation Transfer technology) process is highly reproducible.However,attentionmustbepaidtothefollowingtreatmentattributestoensurebestresults:

• Tubes:Atpresent,therecommendedtubevesselsarethe0.65mlBioruptor®Microtubes(CatNo.WA-005-0500).Payattentionnottodamagethecapwhenclosingthetubessincethiscouldaltersonicationresults.

• Sample volume:Therecommendedvolumeofthe0.65mlBioruptor®Microtubes(CatNo.WA-005-0500)is100µl.Whenusinglowervolumes(e.g.≤50µl),lessreproducibleresultsmaybeobserved 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 largerpeaks.

• Sample concentration:DiagenoderecommendsusingDNAconcentrationrangingbetween1and20ng/µl(10ng/µlrecommended).Usinglargerconcentration(e.g.50-100ng/µl)mayresultinbroaderpeaksorvariablepeakdistribution.

• Sample preparation:Sampleviscositymayhaveamajorimpactonsonicationresults.CarefulresuspensionofDNAsampleisstronglyrecommendedbeforesonicationprocessing.Multiplepipettingandgentlevortexingfollowedbyashortcentrifugationtorecoversamplevolumeatthebottomofthetubeisthereforestronglyrecommended.StoringDNAsamplesoniceduring5-10minutesbeforesonicationhasalsobeenshowntoimprovereproducibility.

• DNA quality: DNA quality and quantity must be considered carefully since bad quality andquantityDNAmayhaveseveralimpactsonsonicationandNext-Gensequencingdownstreamapplications.First,DNAcontamination(e.g.fromsuperfluousnucleicacidssuchasRNA,smallnucleicacidfragments,excessproteins,orothercontaminatingmaterials)mayinterferewithDNA measurement method leading to incorrect DNA quantitation thus. Also contaminatingRNA in genomic DNA preparation might generate a biased fragment distribution profile onmicrofluidics-basedplatform(e.g.AgilentBioanalyzer)oraltersonicationeffiency.

Therefore it is highly recommended to use only high quality DNA when sonicating DNA forNext-Gensequencing librarypreparation.TheDNAsample tobeprocessedshouldbehighlypure,havinganOD260/280ratioofbetween1.8and2.0,andshouldbeas intactaspossible.DNAextractedusingstandardtechniques(e.g.ProteinaseKdigested,doublephenol/chloformextraction, ethanol precipitated, treatment with RNase-DNase free enzymatic digestion toremovecontaminantRNA)orcommercialspin-columnbasedkitsarerecommended.

• Water temperature:PropagationofultrasoundinaliquidunavoidablyproducesheatthatcanultimatelyalterDNAsample(e.g.bythermaldenaturation).Toensurethebestpreservationofthesample,itisrecommendedtostartthesonicationprocesswithcoldwaterinthewaterbath.Duringsonication,especiallywhendoinglongsonicationruns,thetemperaturemustalsobecontrolled.Thisisobtainedbytheautomatictemperaturecontrol.

Note: Thepermanent installationof theBioruptor®Pico inacold room ispossible,althoughnotsufficienttoavoidthetemperatureincreaseduetosonication.

• Automatic temperature control:Arecirculatingwatercoolerisusedtoguaranteetheautomatictemperaturecontrolofthewaterbathduringthewholesonicationprocess.Thiswatercooler(Cat.No.BioAcc-cool)producesaregularwaterflowwithaconstantwater level in thetank.

www.diagenode.com

Anadditionalregulatingvalve(SingleCycleValve,Cat.No.B02020004)ensuresthatwaterwillonlybereplacedduringtheoffcycletoavoidanyinterferencebetweenthewaterflowandthesonicationprocess.

• Sonication time:Minoradjustmentsincyclenumbermaybemadetooptimizeresultsforvarioussampletypesandconcentrations.Cyclenumberlistedaboveisarecommendedguideline.Actualresultsmayvarydependingontheamountandtypeofstartingmaterial,concentration,viscosityand/orplastictubes.Diagenoderecommendssettingupatimedoseresponseexperimentfordeterminingappropriatecyclenumber.Largerlengthstartingmaterial(e.g.totalgenomicDNA)andhigherconcentrationmayrequirealongerdosetoensureahomogeneousshearingresult.

• Water bath:ThesonicationwaterbathisacriticalcomponentoftheBioruptor®Picosonicationsystem.

1. Water purity: Contaminants such as algae and particules may alter the ultrasonic wavespropagation,resultinginbroadersizedistributionorlargerpeaks.Bathwatershouldbepuredistilled water,changedregularly(atleastonceperweek).

2. Water bath maintenance: The water bath metal surface is fragile and requires a carefulmaintenance.Useonlysoftspongetoremovetraces.Neverusescratchscrubspongesincethiswouldaltertheultrasonicwaveemittersurface.

3. Water type:Distilled water

Supplementary Data:

Pleasenotethattherearethreemainsourcesofvariationinbothpeakbase-pairsizeanddistribution:

1) ThephysicalprocessofDNA fragmentationmightnotbeentirely random inAT-orGC-richregions.

2) The analytical process to determine fragment size has inherent variances (for example, gelelectrophoresisandmicrofluidics-basedplatform).Therefore,fragmentdistributionsandpeakvalues,even fromtechnical replicates,maynotappear identical. If theshearedDNAsamplewillberesinorcolumnpurifiedorconcentratedpriortoanalysis,pleaseremembertotakeoutanaliquot foruseascontrolprior to thatstep.Columnpurificationandconcentrationof theshearedDNAwillgenerateabiasedfragmentdistributionprofileduetotheinherentgreaterlossofthesmallerDNAfragments.

3) RNA contamination in genomic DNA preparation should be carefully removed using RNase-DNasefreeenzymaticdigestionsincetheymightgenerateabiasedfragmentdistributionprofileonmicrofluidics-basedplatform(e.g.AgilentBioanalyzer)oraltersonicationeffiency.

Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl...

Documents

PRECISION LIQUID HANDLING - Microlab Tech · 2010-06-22 · MICROLAB ® Robotic ... 55019-09 100 µL 200 µL 21 and 22 55019-11 200 µL 200 µL 21 and 22 55019-13 250 µL 300 µL

Practical 3. Analyzing DNA fragments · the components of your digests on ice ... 6 12 µl 2 µl 2 µl 2 µl 1 µl ... Flick the tube with your finger to mix well and spin for 5 sec

Supporting Information Boosting CRISPR/Cas9 …...Bsa I (10 U/µl, NEB) 0.5 µl T7 DNA Ligase (3000 U/µl, NEB) 0.5 µl Total Volume 20 µl For GG assembly reactions to construct PTG1-PTG8,

ab207201 Total CREB Transcription Factor Assay Kit ......WI-38 (Forskolin) nuclear extract (2.5 µg/µL) 40 µL 200 µL -80°C -80°C Dithiothreitol (DTT) 100 µL 500 µL -20°C -20°C

Fine Care Biosystems - ENDO...VARIABLE 0.1 - 2.5 µl 0.5 - 10 µl 2 - 20 µl FIXED 2.5, 5, 10 µl MUL TI 0.5 - 10 µl VARIABLE 5 - 50 µl FIXED 20, 25 µl MULTI 5 - 50 µl VARIABLE

Electromagnetic shearing machine

Overview - European Bioanalysis Forum...Sample prep PPT 50 µl plasma + 300 µl CH 3CN dilution 100 µl SN + 300 µl H 20 Injection volume 0.5, 1 and 2 µl (with 2 µl sample loop)

AUTOMATED LIQUID HANDLING · MICROLAB M Volume 10 µL 25 µL 50 µL 100 µL 250 µL 500 µL 1 mL 2.5 mL 5 mL 10 mL 25 mL Model 1701 1702 1705 1710 1725 1750 1001 1002 1005 1010 1025

Stepper · 2020. 1. 3. · Aufnahme-Volumen ml Volume prélevé ml 0.5 2.5 12.5 25 Delivery volume µl Dispensier-Volumen µl Volume distribué µl Volumen de dispensacion µl 1020304050

1.Set up 110 µl mix for each primer/DNA combo on ice! 1.1.1 µl 100x F primer (1 pMol/µl = 1µM final []) 2.1.1 µl 100x R primer 3.11 µl 10x PCR buffer 4.2.2

20202017 ALEXMK2 Quick Guide A4...ALEX2 Stop Solution 8 Min 100 µL 400 µL 100 µL 500 µL 500 µL 500 µL 500 µL 500 µL Title 20202017_ALEXMK2_Quick_Guide_A4 Created Date 2/17/2020

PapilloCheck - GBO · Steril, DNase içermeyen mikropipet filtre ucu4 0,5 µL - 10 µL 0,5 µL - 20 µL 10 µL - 100µL 10µL - 200 µL 100 µL - 1000 µL 765 288 774 288 772 288

VIDAS® ASSAYS - PRACTICAL INFORMATION…شخصات... · AFP 100 µL E2II 200 µL AMH 200 µL PRL 200 µL LH 200 µL FSH 200 µL PRG 200 µL CEAS 200 µL 199 200 µL 125 200 µL

Combination of epT.I.P.S.® with Eppendorf Pipettes...pearl white 0.5 – 100 µL light yellow 2 – 20 µL yellow 2 – 100 µL yellow 2 – 200 µL yellow 20 – 300 µL orange 50

Transferpette ¡Primicia mundial! Ergonomía: comprobada y ... · 0,5 - 10 µl, 2 - 20 µl, 20 - 200 µl, 100 - 1000 µl, 0,5 - 5 ml – Aparatos multicanal: ... do es un principio

Managing Degloving & Shearing Injuries Degloving and shearing

SofTouch...8707-06XX 8-Channel 5 - 100 µl1 µl 200 µl, 300 µl 8707-07XX 8-Channel 25 - 250 µl5 µl 300 µl 8707-08XX 8-Channel 50 - 1200 µl 10 µl 1200 µl 8707-09XX 12-Channel

HYDRAULIC SHEARING

KAPA RNA HyperPrep Kit Roche Platforms · 2019. 9. 20. · KAPA HiFi HotStart ReadyMix (2X) 300 µL 300 µL 25 µL 780 µL 50 µL 1 mL ... and is the polymerase of choice for NGS

AriaMx Real-Time PCR Software...Primer (200-600 nM) X µL X µL Probe (150-600 nM) None X µL Template X µL X µL DW Up to 20 µL Up to 20 µL AriaMxリアルタイムPCRシステムの反応液量は10