1.Obtainthelast2mmoftailandplacedirectlyinto200ul1XPCRBufferwithNonionicDetergents(PBND)ina1.5mlmicrofugetube.(TailscanbestoredatfrozeninPBSorPBNDuntiluse.)2.Add1.2ulProKsolution(Ihan"tseenanyproblemwithupto5ulProKandtaildegredationisslightlyfaster)toeachsampleandplaceina55ocbath.3.Incubateat55ocwithoccasionalvortexinguntiltissueisdegraded(1-3hours).4.Heatsamplesto95ocfor10min.inPCRmachineorbyboilingtoinactivateProK.5.Add2ul(note)processedTailDNA/50ulPCRreaction.note:thisvolumewillvarydependingonyourparticularPCRprimers,theirTmetc....*Adaptedfrom:PerkinElmerCetus:Amplifications:Vol.#2PEC1989;pp1-3. GenericPCRConditions2ul(variable)tailDNA:donotvortex,avoidsedimentintube.2uMfinalPrimers0.025U/ulTaq100-200uMdNTP"s1XPECPCRBufferw/outMgCl21.5mMMgCl2H2Otofinaltotalvolumeof50ul Reagents1)(PBND)PCRBufferwNonionicDetergents:For500mlfinalvolume:50mMKCl1.87gKCl10mMTris-HCl(pH8.3)##5ml1MTris-HClStock2.5mMMgCl2-6H2O255mgMgCl2-6H2O0.1mg/mlgelatin50mggelatin0.45%v/vNonidetP40(NP40)2.25mlNP400.45%v/vTween202.25mlTween20PCRProtocol
Note:LysisbufferfortailprepisPCRcompatIBLe(notusuallycleanenoughforrestrictiondigests).