Oakley_PCR_protocol

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1、OakleyLabFusionPCRProtocolP1P2P41.HistoneH13’UTRP3P5P6AmplificationofgenomicDNAwithP1andP3AmplificationofgenomicPrimersDNAwithP4andP6PrimersPrimersandnucleotidesareremoved.2.P2mRFPAfpyrGP5FusionPCR.mRFP--AfpyrGwaspreviouslyamplifiedfromaclonedcassette.Thethreefragmentsaremixedandampl

2、ifiedwithnestedprimers(P2andP5)3’UTRHistoneH1mRFPAfpyrGLinearfragmentsuitablefortransformationafterremovalofprimersandnucleotides.ThefigureaboveisaschematicdiagramforgenerationofalinearfragmenttotagtheC-terminusofHistoneH1withmonomericredfluorescentprotein(mRFP).Thesameapproachcanbeuse

3、dtotagtheC-terminiofgeneswithGFPandotherfluorescentmoetiesorepitopetags.Itcanalsobeusedtoreplacegenesortoreplacethepromot-ersofgeneswithregulatablepromoterssuchasalcA.Thefollowingprocedureisveryrobustandreliableinourhands.ItconsistentlyproducesfusionPCRproductsthatareadequatelypuretob

4、eusedwithoutbandpurificationtotransformnkuAdeletionstrains.Abenefitoverapreviouslypublishedprocedure(Yangetal.,2004,Eukaryot.Cell3:1359-1362)isthattheoverhangsthatallowfusionPCRareontheprimersthatareusedtoamplifythegenomicfragments(primersP3andP4specifically).ThissavestwoPage1OakleyLabF

5、usionPCRProtocolprimersfromthepreviousprocedureandallowsthecassette(mRFP--AfpyrGinthiscase)tobeamplifiedonce,frozenandaliquotsusedinmanyfusionPCRreactions.Firstoneneedstoordersixprimers(P1-P6)asshowninstepone.Wenormallyusehomologousflankingregionsabout1000bpinlengthsoP2andP3wouldnormal

6、lybeabout1000bpapartaswouldP4andP5.P1isjust“outside”P2andP6isjust“out-side”P5.P1,P2,P5,andP6arenormallyabout18-21bpinlengthandhaveameltingtemperatureofabout60ºCusingthefollowingformulaformeltingtemperature.Tm=[(C+G)X4]+[(A+T)X2]whereC=thenumberofC’sintheprimer,G=thenumberofG’sinthepr

7、imer,etc.TmisofcourseinºC.Longerprimersdonotworkbetterandof-tenworkworsethanprimersofthislength.PrimersP3andP4arelonger.P3UsingP3asanexample:Thissegmentisidenticaltotheantisenseofthe3’endoftheHistoneH1codingregion.Itis18-20bpinlengthandhasameltingtemperatureofapproximately60ºC.Thisse

8、gmentisident