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1、3페이지1/7------------------------------------------------------------------------------------------------------------SectionIRNAligationof3`linker.1.Theuseoftheadenylylatedoligo“modban”(availablefromIDTDNATechnologies,idtdna.com)precludtheneedtoincludeATPintheligation,andhencehelpspreventc
2、ircularizationoftheRNA.Setupthefollowing20µlligationreaction:13µlwatercontaining5-50microgramsoftotalRNA2µl10XT4RNAligasebuffer2µlDMSO1µl100µMRNA3’linkeroligonucleotide,“modban”AMP-5’p-5’p/CTGTAGGCACCATCAATdi-deoxyC-3’.2µlT4RNAligase(20Units,Amersham)Incubateat37oCfor1hour.Stoptheligatio
3、nbytheadditionof20µlofdenaturinggelsamplebuffer.Thestoppedligationreactioncanbestoredat–20oC,orprocessedimmediatelybygelelectrophoresis.Thereactioncanalsobeextractedwithphenol,precipitatedwithethanol,andthenredissolvedinsamplebufferpriortogelelectrophoresis.***10XT4RNAligasebuffer:500mMT
4、ris-HClpH7.5,100mMMgCl,100mMDTT;10mMATP,600µg/mlBSA2***SectionII.Gelpurificationof(RNA-to-3’linker)ligationproduct.1.Preparea12.5%acrylamide8Mureagel,4cmwideby1.5mmthickby8cmlong,usingacombwithacmwidelane.(TheBio-RadProteanIIminigelsystemorequivalentisrecommended).Accordingly,mix:7.5mlSe
5、quagelConcentrate(NationalDiagnostics)6mlSequagelDiluent1.5mlSequagelBufferDe-gasbyvacuumfor1minute,thenadd:150µl10%ammoniumpersulfate3.5µlTEMEDMixgentlyandpourthegel.2.Heatthesampleto65oCfor5minutes,chillthetubeonice,andloadontothegel.Includeamarkerlanewith15microgramsofa40-mersingle-st
6、randedDNAoligonucleotide.Electrophoreseat18volts/cm(150voltsfora8cmgel)untilthefasterdyereachesthebottomofthegel.3.Visualizethe40ntmarkerbyUVshadowing,andmarkitsposition.Thisistheexpectedpositionofligatioproduct(18ntModbanplus~22ntsmallRNA).ElutetheligatedRNA(40nt)fromtheacrylamidegelsli
7、casdescribedbelow,(butincludeanextra3-4mmofgelaboveandbelowthelabeledbandofligationproduct).5.Placethegelsliceinaclean1.5mlmicrocentrifugetubeandhomogenizeitwiththesmallendofa1000µplasticpipettetipthatyouhadmeltedshuttoarounded,roughlysphericalshapeatthetipbybrieflypassin
