ThefollowingcolonyPCRprotocolhasbeendesignedtobeperformedinindividualreactiontubes.Weusuallytestthreecoloniesfromeachtransformationalongwithawild-typecontrol.AseriesoffivedifferentPCRtestsareperformedoneachcolonytoconfirmbothofthenovelrecombinationjunctions.

1.Clonalpurification

-PickthreecoloniesfromatransformationplateandstreakeachoneouttosinglecoloniesonseparateG418containingplates(200mg/l).-Incubateat30°Cfortwotothreedays.*Thisstepreducesbackgroundbyeliminatingabortedtransformants.

2.Zymolyasetreatment

-Transferasmallportionofawell-isolatedcolony(~1mm)into50µlsolutioncontaining60U/mlofZymolyase(ThissolutionisgeneratedbyresUSPending6mgof100TZymolyasein10mlsofwater.Orderinginformation:SeikagakuCorp.,#120493-1).

-Sterileyellowtipsworkgreatforthistask.-Repeatthisstepforeachofthe3isolatesandthewild-typecontrol.-IncubatetheZymolyasesolutionsat37°Cfor30minutesfollowedby10minutesat95°C.*ItisnotnecessarytoremovetheZymolyasesolutionbypelletingthecellsbeforethePCR.*TheZymolyasetreatedcellscanbestoredat4°CforseveraldaysbeforebeingusedastemplateforthecolonyPCR.However,fresherisprobablybetter.

*TheamountofcellsthatgetspickedintotheZymolyasedoesnotseemtobetoocritical-similarPCRresultswereobtainedwhen1µl,5µlor10µloftheZymotreatedcellswereusedastemplate.

3.PCRreactions

-Thelyophilizedconfirmationprimers(~5-10nmolesofeacholigonuleotide)shouldberesuspendedin750µlofTE(finalconcentrationof~10µM).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.Eachofthe20tubesshouldcontain10µloftheprimermix(5µlofeachprimer).-Addthe5µloftheZymotreatedcellstoeachofthe20PCRreactions.Forexample,add5µloftheZymosolutionfromisolate#1toPCRtubes1-5,5µloftheZymosolutionfromisolate#2toPCRtubes6-10andsoon.-MakeaPCRmastermixbycombining:638µlwater,110µl10xTaqBuffer,11µlNTP"s,and11µlTaqPolymerase.-Add35µlofthePCRmixtoeachofthe20PCRtubesthatalreadycontainthe15µlofprimersandtemplate.

110µl5µl10xTaqbuffer(seebelow)11µl0.5µl20mMdNTP"s(0.2mM)11µl0.5µlTaqPolymerase(2.5units)638µl22X29µlwater5µl10µMupstreamprimer:A,C,orkanC(1µM)5µl10µMdownstreamprimer:B,kanB,orD(1µM)5µlZymolyasetreatedcells50µltotalvolume

*10xTaqbuffercontains:100mMTris-HCl(pH8.4),500mMKCl,15mMMgCl2.*Thefinalconcentrationsareshowninparentheses.*TheTaqPolymeraseshouldbeaddedlastandPCRmixtureshouldbekeptoniceuntilthePCRisstarted.Alternatively,a"hotstart"canbeperformedbyaddingtheTaqpolymerasetotheindividualtubesafterthePCRmixtureshavebeenheatedto94°C.HotstartimprovesthePCRresultsbutthisstepislaborintensiveandwehavenotfoundittobenecessary.

4.PCRconditions:

5.Agarosegelelectrophoresis

-Add10µl6xloADIngbuffer(30%glycerol,50mMEDTA,0.25%bromophenolblue)tothe50µlPCRreaction.-Load10µlona1.5%agarosegel.