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TwistAmp®DNAamplificationkitsCOMBINEDINSTRUCTIONMANUALFREQUENTLYASKEDQUESTIONS®process.Inparticularthosesystemsemployingthe5’to3’nucleaseactivityofpolymerasescannotbeusedwiththeTwistAmp®systemassuchenzymaticactivityisfundamentallyincompatiblewiththeRPAbiochemistry.HowdoIdesignRPAprimers?GoodRPAprimersareidentifiedbyascreeningprocessdescribedindetailintheAppendixat®reactions?YES.Itispossibletoperformmorethanoneamplificationreactionsimultaneouslyinthesametube.However,notallprimerpaircombinationswillworkequallywellwitheachotherinmultiplexingandthisformatthereforerequirescarefulprimerdesign.Notethatthetotalamount(nmols)ofoligonucleotideinthereactionshouldnotsignificantlyexceedthatstatedintheprotocol;ifmorethantwoamplificationprimersareusedinasinglereaction,thenthemaximumamountofprimerhastobedividedbetweenalltheoligonucleotidespresent.Monitoringmultipleamplificationeventsatthesametimemightalsorequiredifferentprobes,andlimitationsonboth,thedetectionequipmentandtheavailabilityofcompatiblefluorophores,havetobeborneinmind.HowmuchofmyprimersdoIhavetouse?TherecommendedconcentrationofprimersintheTwistAmp®Basicreactionis480nMeach.IntheTwistAmp®exo,TwistAmp®fpg,andTwistAmp®nfokitstherecommendedconcentrationof(from200nMto600nMeach)canbeemployedtoidentifytheoptimalconcentrationconditionsforagivenprimerpair.HowdoIreconstitutealyophilisedprobe?Pleasefollowtheoigonucleotidemanufacturer’sinstructionsforthereconstitutionandstorageoftheprobes.TypicallythetubecontainingthelyophilisedoligonucleotidewillbespunbrieflytocollecttheDNAatthebottomofthetubeandanappropriatevolumeofT0.1Ebuffer(10mMTris-HCIpH8,0.1mMEDTA)willbeaddedtoprepareastocksolutionof100μm.Allowthesolutiontostandfor10minutesatroomtemperatureandmix(vortex)for10seconds.Reconstitutedoligonucleotideprobesaretypicallystoredat-20°CforthelongtermHowmuchofmyprobedoIhavetouse?Therecommendedconcentrationofprobeinthereactionis120nM.However,someTwistAmp®reactionsbenefitfrombeingusedwithslightlydifferentamountsofprobe.Testingdifferentconcentrationsofprobe(from50nMto150nM)willhelptooptimisetheperformanceofprobebaseddetectionformatsofagivenassay.CanIuseTwistAmp®reactionsforthequantificationoftemplate?YES.Theonsettimeofdetectableamplificationforagivenassaywilldependontheamountofstartingtemplatematerial–themoretemplatecopiestherearetostartwith,theearlierthedetectiontimewillbe.However,exploitingthis‘time-based’quantificationdemandsacarefulexperimentalsetupensuringsimultaneousinitiationofcomparedreactions(e.g.through‘Magnesiumstart’).Theresolutionofquantificationisaidedbyarelatively‘slow’amplificationreaction.Strategiestoslowtherateofamplification(includingthedesignofsuitableprimers)arediscussedintheAppendixat®Basic,nfoorfpgreactions.HoweverthereactioncomponentsareactivatedassoonasMagnesiumisadded.BypipettingMagnesiumAcetateintothereactionlast,whichwerecommend,forexamplebyaddingittothelidsofstripsandspinningitintothereactions,youensurethatreactionsstartsimultaneouslyandyouminimisetheriskofcrosscontaminationorRPAproductsbeingproducedinanyreactionresiduesleftinyourtips.NO,ifyouareusingTwistAmp®exoorTwistAmp®exoRTreactions.InthepresenceofMg,theexonucleasecanattacktheprimersandprobesbeforetheyareprotectedbytherecombinaseandsingle-strandedbindingproteins.Thismaycauseahighfluorescencebaselineinyourreactions.CanImakeamaster-mix?YES.Ifyouwishtosetupmultiplereactions,youcanmakeamastermix.IfyouarescreeningdifferentDNAs,theresuspensionbuffer,primersandprobeifusedcanallbemixedtogetherandaddedtofreezedriedreactionstoresuspendthem.DifferentDNAscanthenbeaddedtoreactionsbeforetheyarestartedwithMgAcasusual.Ifyouareperformingaprimerscreen,itispossibletomakeamastermixwiththeresuspensionbuffer,templateDNA,probe(ifused)andoneoftheprimers.Thisshouldbealiquottedinto1.5mltubesandthevariableprimersadded.Onlyoncebothprimersarepresentshouldthefreezedriedreactionsberesuspendedortheformationofrecombinationfilamentswillbebiasedtowardsthefirstoligonucleotideadded.