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1、PrimerDesignProtocolauthor:WendyPhillipsDate:1998Annealingtemperature,GCcontent,lengthIfyouaredesigningtwoprimerstobeusedwitheachother,orifyouaredesigningaprimertobeusedwithanexistingprimer,trytodesignthemsotheywillhaveasimilarannealingtemperature.Therearemanyformulastopredictannealingtemps.Som
2、emaygivebetterestimatesthanothers,butintheenditallneedstobedeterminedempiricallyanyway.ThatiswhyIusethesimplestformula.AnAorT=2°,aGorC=4°.CountupallAsandTsandmultiplyby2°.CountupallGsandCsandmultiplyby4°.Addupthosetwoproductsandyou’llgetapredictedannealingtemp.Thisworksforacertainsizerange–Idon
3、’tknowwhatitis–butyouraverageprimershouldfallinthatrange.Ifyouhavedegeneratebasesinyourprimer,youshouldeitherputinanaveragevalueforthepossiblenucleotides(ex.IfitcouldbeAorG,use4°,ifitcouldbeA,T,orGuse2.7°)oryoucoulddeterminethelowestandhighestpossibletempsforthatprimer,i.e.onetimeassumeanydegen
4、eratepositionisthelowesttempchoiceandonetimeassumeanydegeneratepositionisthehighesttempchoice.Noteifyougowiththeaveragingoption,you’llprobablywanttostartoutusingthatprimerafewdegreeslowerthanwhatyoucalculatedtobesureithasachancetostick.Ithinktheidealprimerisonewith50%ATand50%GC.HavinglotsofGCsm
5、ayseemgoodbecausetheyaresticky,butifyouhavetoomanyyouareincreasingthelikelihoodthatitwillsticksomewhereitshouldn’tbecauseitmaymaketheAandTpositionslessmeaningful.IfyouhavetoomanyAsandTs,youmightnotgetasgoodofastick.Ifatallpossible,Idefinitelytrytokeeptheratiointhe60:40rangeeitherway.Ilikeprimer
6、s18-20ntsthemost.Myidealprimerisa20-merwith10ATs,10GCs,annealingtemp60°.3’endstickinessAlongthosesamelines,youwanttopayattentiontotheGCcontentofthe3’end.The3’endismuchmoreimportantthanthe5’end.Ifthe5’enddoesn’tstickperfectly,theprimercanstillwork.Ifthe3’enddoesn’tstick,theprimerwillnotwork.Some
7、peoplereallyliketohavethelasttwo(3’most)ntsbeGorCtogivetheprimermore3’endstick.ItrytoendwithoneGorCifpossible,butifIcan’t,Ican’t.Bewarethough,youdon’twanttoomanyGsorCsatthe3’end–4outof5ofthelastntsareGorC–thatisgettingtobetoomany.
