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1、SingletubeconfirmationPCRprotocolThefollowingcolonyPCRprotocolhasbeendesignedtobeperformedinindividualreactiontubes.Weusuallytestthreecoloniesfromeachtransformationalongwithawild-typecontrol.AseriesoffivedifferentPCRtestsareperformedoneachcolonytoconfirmbothofthenovelrecombinationjunc
2、tions.1.Clonalpurification-PickthreecoloniesfromatransformationplateandstreakeachoneouttosinglecoloniesonseparateG418containingplates(200mg/l).-Incubateat30°Cfortwotothreedays.*Thisstepreducesbackgroundbyeliminatingabortedtransformants.2.Zymolyasetreatment-Transferasmallportionofawell
3、-isolatedcolony(~1mm)into50μlsolutioncontaining60U/mlofZymolyase(Thissolutionisgeneratedbyresuspending6mgof100TZymolyasein10mlsofwater.Orderinginformation:SeikagakuCorp.,#120493-1).-Sterileyellowtipsworkgreatforthistask.-Repeatthisstepforeachofthe3isolatesandthewild-typecontrol.-Incub
4、atetheZymolyasesolutionsat37°Cfor30minutesfollowedby10minutesat95°C.*ItisnotnecessarytoremovetheZymolyasesolutionbypelletingthecellsbeforethePCR.*TheZymolyasetreatedcellscanbestoredat4°CforseveraldaysbeforebeingusedastemplateforthecolonyPCR.However,fresherisprobablybetter.*Theamountof
5、cellsthatgetspickedintotheZymolyasedoesnotseemtobetoocritical-similarPCRresultswereobtainedwhen1μl,5μlor10μloftheZymotreatedcellswereusedastemplate.3.PCRreactions-Thelyophilizedconfirmationprimers(~5-10nmolesofeacholigonuleotide)shouldberesuspendedin750μlofTE(finalconcentrationof~10μM
6、).Weuse5μlofeachprimerin50μlPCRreactions(~1μMfinalprimerconcentration).-Label20thin-wallPCRtubes(5foreachisolate)andaddthefollowingprimerpairs:A-B,A-kanB,C-D,kanC-D,andA-D.Tubes1-5areforisolate#1,6-10areforisolate#2,...,andtubes16-20areforthewild-typecontrol.Eachofthe20tubesshouldcont
7、ain10μloftheprimermix(5μlofeachprimer).-Addthe5μloftheZymotreatedcellstoeachofthe20PCRreactions.Forexample,add5μloftheZymosolutionfromisolate#1toPCRtubes1-5,5μloftheZymosolutionfromisolate#2toPCRtubes6-10andsoon.-MakeaPCRmastermixbycombining:638μlwater,110μl10xTaqBuffer,11μlNTP's,and1
8、1μlTa
