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1、McManus,M.T.SharpLab,MIT-03/01/03SmallRNANorthernBlotsSmallRNANorthernblotsareactuallyquiteeasy,onceyouaresetupforit.Ingettingsetupforit,IrecommendperformingacontrolblotinwhichDNAoligosareloadedontothegel(insteadofRNA).Seemynoteoncontrolsbelow.IngredientsTobegin,makesuretogetther
2、ightingredients:smallRNANorthernblottingcanbeespeciallysensitivetothemembraneandhybconditions,andthatiswhyyouwillwanttobeginwithacontrolblot.Forahybridizationbuffer,IprefertouseAmbion’sExpressHyb,althoughhomemadepreparations(5%SDS,200mMNa2PO4pH7.0)workwellalso.IprefertouseHybondN
3、+membrane(Amersham)becauseithasprovenconsistentlysuccessfulovermultiplebatches.Othermembranes,suchasGenescreenPlus(NEN)alsowork,justbesuretorunthebelowcontrolwitheverynewmembranebatch.U6-drivensmallRNAsandsomemicroRNAs(suchaslet-7)arereallyabundant,andcanbevisualizedquitereadilyw
4、ithjustafewhoursexposure.TheBlot:Using~17cmwide,~30cmlongglassplates,poura1.5mMthick,10%DNAsequencingPAGEgel.Iprefertousea10-12toothcomb,whichwillallow60ugormoreRNAtobeloadedperlane.Itypicallypre-runthegelsuntilthetemperatureis~50°C,ataroundaconstant40mAorso,dependingonyourpowers
5、upply.RunthegeluntiltheBPBhitsthebottomofthegel,asyoursmallRNAshouldbeaboutmidwaybetweentheXCandBPBdyes.Onasemi-drytransferapparatus,transfertheRNAsfromyourgeltothemembrane.IuseaBioRadtransblotapparatusrunat250mAforabout3hours.Afterthetransfer,crosslinkthemembraneat1200mjoules.Du
6、ringthetransfer,prepareyourprobeby5’-endlabelinga25-50pmolesDNAoligoina~132hourkinasereaction.RemovefreePbyETOHpptordesaltingcolumn.Aftera~30minuteequilibrationofyourmembraneinthehybbuffer,addfreshhybbuffer(containingyourlabeledprobe)andhybridizebetween4hoursandovernightat37°C.Iu
7、suallydothisin50mldisposableconicaltubes,andmybuffervolumeis5ml.Forthewashes,Isuggestgraduallysteppinginthestringency.Trystartingwith3washes2xSSC,0.1%SDSatRT,exposing,andifthatisnotcleanenough,tryreducingto0.5X,0.1%SDS,orlowerSSCconcentrations.Ifbackgroundisaproblemina1-2dayexpos
8、ure,somethingiswrong,sotroubleshootyourb
