Single tube confirmation PCR protocol

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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