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Epigentek/EpiQuik Circulating Cell-Free DNA (cfDNA) Isolation Easy Kit/P-1065-25/25 isolations

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InputType:UnisolatedSamplesResearchArea:DNADamage&Repair,DNAMethylation,NextGenSequencingTargetApplication:SampleIsolationVesselFormat:Plate/Tubes100%Guarantee:6monthsProductOverviewThe EpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit utilizesmagneticbeadsbasedsize-fractionationtechnologytoisolatecirculatingcell-freeDNA(cfDNA)fromplasma/serumsamplesinasimpleandfastmanner.TheisolatedcfDNAcanbedirectlyusedforrealtime-PCRandDNAlibrarypreparationsuitablefornextgenerationsequencing. Thekithasthefollowingadvantages:Fastandstraightforwardprocedurecanbefinishedwithin1hour.Usesinnovativemagneticbeadbasedsize-fractionationtechnologyforisolationofcfDNAfromplasma/seruminasimpleandconvenientmanner.TheisolatedDNAcanbedirectlyusedforbothqPCRandNGSDNAlibrarypreparation.Efficientremovalofproteins,salts,nucleases,PCRinhibitingsubstances,andotherimpuritiessuchaspolysaccharides,polyphenolsandlipids.SensitiveandefficientDNAcaptureenablessuccessfulisolationwithhighrecovery(>80%ofinputmononucleosomalDNA),evenwhenthequantitiesofstartingmaterialarelimited(aslowas0.2ml). BackgroundInformationGeneticandepigeneticanalysisofcirculatingcell-freeDNA(cfDNA)inplasma/serumorotherbodyfluidsprovidesuniqueopportunitiesforearlydetectionofawiderangeofclinicaldisorderssuchascancer,autoimmunedisease,infectionandfetaldisorders.ItwasdemonstratedthatcfDNAofclinicalimportanceoccurspredominantlyasfragmentsofapproximately170basesfrommononucleosomeswithasmallerproportionasfragmentsof360basesfromdi-nucleosomes[1,2].Suchnucleosomalcomplexesarereleasedintobloodcirculationduringapoptoticcelldeathandwillbeincreasedundervariouspathologicalcircumstancessuchasinflammation,pulmonaryembolism,autoimmunedisease,andcancer[3,4].ItisalsoshownthatcfDNAfromnucleosomalcomplexesinserumandplasmaissmallsizefragmentDNA(170-500bps)andusingsuchcfDNAforgeneticorepigeneticanalysisprovidesbetterandmoreaccurateidentificationofphysiologicalandpathologicalstatus[5]. Principle&ProcedureTheEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKitcontainsallcomponentswhichhavebeenoptimizedforthesimpleandrapidisolationofsmallsizecfDNAfromplasma/serum.ThecirculatingnucleosomalcomplexesarefirstdigestedandDNAisthenenzymaticallyreleased.ThecfDNAisefficientlycapturedviasize-fractionationmagneticbeads(cfDNACaptureBeads)byapplyingthebeadstoamagneticfield (EpiMag™HT(96-Well)MagneticSeparator orsimilar).ThecapturedcfDNAispurifiedbysimplywashingthe beads.ThepurifiedcfDNAisthenelutedfromthebeadsforimmediateuseorstorage.StartingMaterialsBothfreshandfrozenplasma/serumfromvarioussourcescanbeused.However,freshplasma/serumwillgenerallygivehigherDNAyieldsthanfrozen.Theinputvolumeofplasma/serumcanbefrom0.1-1mlwiththestandardvolumeof0.5mlpersample.Ifserumsampleisused,theserumshouldbepreparedwithin6hoursafterblooddraw,sincelysisofperipheralbloodlymphocytesmaycauseanartificialincreaseintheamountofDNAduringserumseparation. References:1.JahrSetal:CancerRes.2001,61:1659-16652.SuzukiNetal:ClinChimActa.2008,387:55-583.HoldenriederSetal:CritRevClinLabSci.2009,46:1-244.SchwarzenbachHetal:NatRevCancer.2011,11:426-437 5.ChanKCAetal:ClinicalChem.2004,50:88-92 Fig.1. WorkflowofEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit.Fig.2. HighrecoveryofcfDNA:DifferentamountsofHeLamononucleosomeswerespikedinto0.5mlofplasmathenisolatedusingtheEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit.TheisolatedDNAwasfluorescentlyquantified. Fig3. HighrecoveryofcfDNAconfirmedbybioanalyzeranalysis: 500ngofHelapolynucleosome(around3000bps)and500ngofmononucleosome(around170bps)weresimultaneouslyspikedinto0.5mlplasma andthenisolatedandsize-selected. Fig4. BioanalyzertraceofDNAlibrarypreparedfrom20ngofpurifiedmononucleosomesisolatedfromplasmaspikedwithbothmono-andpoly-nucleosomes.Librarypeaksize:350bps. ProductComponentsProteinaseKcfDNACaptureEnhancercfDNACaptureBeadsCaptureBufferElutionBufferUserGuide

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