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1、菌落pcr资料（ColonyPCRdata）ColonyPCR(Colony,PCR)methodColonyPCR(Colony,PCR)doesnotneedtoextractgenomicDNA,withoutenzymecuttingidentification,butdirectlyusestheDNAexposedafterbacterialpyrolysisastemplateforPCRamplification,withlesstimeandeffort.Universalprimersarer
2、ecommendedforuseonvectors.ThismethodisusuallyusedforrecombinantscreeningorDNAsequencinganalysis.ThefinalsizeofthePCRproductisthesizeoftheinsertionfragmentbetweenthevectorsandtheuniversalprimers.Concretemethod:Preparationofmixturesof1andPCRTaqbuffer(10x)20ULDN
3、TP(2.5,mM)5ULPrimerForward(50mM)10ULPrimerReverse(50mM)10ULDdH2O147,ULTaq(2U/ul)8ULTotal200,ULMixthesolutionstogether.10ULeachtubeispackedina200ULPCRtube.Transformantsontheboardinto2randomlyselected,undernormaltemperature,withatoothpickorgunheadofacellwereste
4、rilized,clickonLBagarplate,makeacopy;andthenstainedwithatoothpickorgunheadonthecellcorrespondingwithPCRmixtureofPCRtubewashingnumber(goodmark,pipeiftheflatpointisNo.1,No.1isalsomarkedonthetube,soastoselecttocloneafterexpandingtotheculture,tightlytube).3.ThePC
5、RmixturemixedwithbacteriawasplacedinaPCRinstrumentandamplifiedaccordingtoconventionalconditions.Didtheelectrophoresistestgetthetargetfragment?.Ifyes,positiveclones.4.Theplatesinoculatedwithcolonieswereincubatedin37incubatorforthenight,andthecolonieswereamplif
6、ied.Thepositivecloneswereselectedforscreeningorculturingthenextday.Step2canalsonotpointboard,directinoculationofshake,andifthemorningtodoPCR,thenintheafternooncanbeimprovedgrain,getrecombinantvector.Note:thedesignofprimersiscritical.Ingeneral,ifdirectedclonin
7、g,theuseofuniversalprimersonthecarriercan;suchaspETseriescanbeusedT7universalprimers.Ifitisanondirectionalclone(suchasasingleenzymecutoraflatendconnection),aprimerwithavectorandaprimeronthetargetgenecanbeeasilyidentified,andtheerrorprobabilityisverylow.ThePCR
8、conditionisclosetothebestselection,whileselectedbacteriashouldnotbetoomuch,otherwisetherewillbenon-specificamplification.ColonyPCRAmplify,SSLP,marker(CER454202),from,51MO5,Agro,streaks.E.
