qPCR文库定量方案优化与操作流程.pdfVIP

NGSLibraryQuantificationProtocol

•MakefreshdilutionsofstandardsolutionsimmediatelybeforeqPCR.Itcanbestoredat4°C

foruptooneday.

•Lowconcentrationstoragebufferincludedinthekitisrecommendedforthedilutionof

librarystandardandunknownlibrarysamp.

•Ensurethatallthereagents(including2Xmastermix,7librarystandardsanddiluted

unknownlibraries)arethoroughlymixedpriortouse.

Step1:DiluteStandardLibraries

•Add49.5µlofstoragebufferto0.5µlof10nMstandardlibrarytemte.Thiswillgivea

concentrationof100pMofthestandard.

•Mixthesamplethoroughlybyvortexingfor15secondsfollowedbybriefspin.

•Add90µlofthestoragebufferto10µlofthedilutedtemtetomakeatitrationcurveofsix

10xserialdilutions.Thiswillgive7controltemtedilutionsintherangeof100-0.0001pM.

•Vortexthedilutiontothoroughlymixthesamp.

Step2:DiluteUnknownLibraries

•EstimatetheconcentrationofunknownlibrarybyOD260reading.Dilutethelibrariesto

approximately10nMinTEbuffer.

•Add998µlofstoragebufferto2µloftheunknownlibrarytemtetomakea500fold

dilution.Thiswillgiveanapproximateconcentrationof20pM.

•Vortexthedilutionthoroughlyfollowedbybriefspinning.

•Add10µlofthedilutedlibraryto90µlofstoragebuffertomakeasecond10-folddilution.

Step3:PrepareReactionMix

Itisimportanttomakeamastermixtominimizepipettingerrors.Themethodheremakesenough

mastermixtofilla96-wellte.Adjustvolumeaccordinglyforyoursamp.

•Preparethereactionmixasfollows:

Consumablereactionmixforonesamplereactionmixfor110samp