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1、primer6.O引物设计程首先,须要知道基因序列，如，我们要设计rat的CK18mRNA检测的引物，我们先到这个网站查找CK18mRNA的基因序列，ZncbiNiimiC4M*rcgr*54StrctGmA6FmGewct&MdcmGn7XlJj*NCBI!“foBMMtf09yhto*Mt%*sy*.MdhM*ro*R*9ccm*Iobcm-：IMc1110yasIRmwt12K三OVUOMMPMCPopSetProoeProtenProtonCU5*sPuQecnPuOCnemCOnPoUrKJPbCnttnrf力/datMmgNCerIGftNC8dMerMtwwe153W011*WW
2、J10GAdcdPMHwEGMOw*nRattusnorvegkuskeratin18(Krt18)1mRNA然因序列号NCBReferenceSemeMM.0639761FASTAor三TWW5a11三三cwJ11affwortl11Wt5InUM1.SMIW3aMUluarX0MRWFiiwMMttinl(Xftl9)BKMaM1.M妙”U_2：661n.aS30?4.1C2：eitM7MeKwitwtucvcuf(IfomaE)1tNCEn*l9KcKma1i*KutAa皿ueh3To”CHruKodayxttSctun*hl.4MBn4wdnr*Ktttv.1C5,ItinBor4lM
3、F,E1.193rrK.9mmTardl*rcauI.Iemin1/18terUe-eh-*adtdaccyrkltcnva0FVS.E)tTW1201百UM现在，往下拉就会看到基因序列tr.fync*msItl-ir/ftMvlar(1.rMe=AAB91db.xref=OuSTS21111.S*ORICDIIccatcettctccctcfttdcctctcca(e&a*taceaccecccc61CXCtCcttCtcecc*etccctccctttctctttctceee，cw，t121ccecctcMCMeCHccacttctatgc(tgct(icctcMrtec“N181MCC贝
4、CtCCCCCUCCtCccctccctCCtCCCtccCCKccctCCCCCMCC241t(cctccaact(*Maa*cct*cUECCaC1.cct301M*caamcc(aaaccaaacsCM*c1*1a*Cc*lk*cccasta3ltetcM*MKttcceeMCCCttCMdacttcaceeatc421MNCtggcctctctttoc*ntctg“,Zm，oCegCSCgtcctgcacat181CBcaatccCetG“c”U3tg1ctt7utcctatgMaccaactcccat541(ccca(t1.ct*ct(MattE“c1cc(cmCtCtCC*tccu
5、cat601caea(tcac1*c*cHc,acgctea(vct(ct(ttca1*a661(aa*aefetCM4MC*Mtee*Mtcctwctcatt*CCNttctcCttc721”WlgatfCtCCCMRCtCMeEwClCNyCKICfttCCftK?81CClCtat3HCCtCMWXcfMgactfWMCtdetCetUC“ca841(1，CaMaCMcaccc,atcaccacCMtct(CC(Mca(c，cd“x901ce*ccttcEccacct(*st(acct(aeteaca961a*aeca4*eaCMCcaocwctctCC*MtCcMcatcacyc102
6、1，hcmctc*ktct(tccttctcatctca*tcftEyCcCtyIOSiM&KCCKcccccocMCaMUCaMCCctet”axH8g4tcacraIIQImat，cccctaccc“ct，BetCC*CCCCCxatttcCtctCa1201ccccc*ctcca*cttccaCK*e*c*ccc1261，M，,c“aCt(tttceacd,accMMttCtydwca1321RWaXcccttgc(ftytecRttyweezam2aIWlaawaaaa&via/将这个序列复制到primer6.0中就可以进行引物设计,我们先打开Peimer6.015, UPgPdeAv
7、ailableAnVfigradraVnihh)Ponthesp11t.DoyouWaIUtodownloaditnow?UPgIadeDeSerdon:UpgradeInChJdeSthefbDowinfeatures Supponforthelatestgcno11ucBabaSeSavailableattheNCBlB1.ASTWrVer SupportforWindows8Forplease5XhttowwwOrElKTbo某OftCorn/ProductgZdEyvVpgradgghtmlKtoctnfornabonISaadabCeat1即/TwwwFPocUjgtgn破解版打开会
8、出现这个对话框，关闭即可FileEditViewAnalyzeToolsOnline点击新建将序列更制至这个对话框NewSequenceSequenceDMnitionJNucleotidesequenceSequenceGCA9611021AAACCAGAJICGATGGAGcAATCCTTGGCtcaatgggcAGCACCTCGGGGMGTGGAGCCCAGATCAGG-jAAACTTCCTTCTGCATctggaatcaGAGCTGGCacGGC1081AGAAGGACAGCGeCAGACCCASAATACGAAgccctcttgAACATeAAGGTCAAOTGA1141GGcGGA
9、GATTGccacctaccGccgcttgctGGGGTGGGGACGATTTCAGTCTC.CGAIZOlCGCCCTGGACTccagcaactCcatgcaaacTgtccagaggACAACTACCCGTAAG-CGT1261GGATGGCAGtgotgtccgAGACCAATGATACCAGAGTTCTGAGGCACTAGGC,AGA1321AGj1.AGGGAJlCCCCTTGGGGACTGJ1.GGGACCMTaJ1.AAGTTTGTCCJUIMAJ1.AA1381kkkkkkAAAAAAAVJMAddICancelIHelp点击AddFoed田TooK0rtnHelpA0/x
10、”|0,51|也匍鼻=Wl-SeuehceInformaflonSearchStatus点击这个图标出现卜面对话框，这里可以选择引物在序列哪些地方设计引物，我一般不更改，也就是引物可以设计在整个序列，你也可查一下序列才几个区，一般引物应当跨两个区。之后点击primerParameters这里可以设置退火温度、引物K度、产物K度等，我一般只是更改产物K度，我一般设为100到300bp,因为假如产物太大，在染料法中会影响扩增效率,之后点击Search之后出现下面对话框SearchCompletedSequencesProcessed:1EIapsedTime00:00:00点击OK出现下面引物序列
11、这个时候引物设计完成，我们须要对引物进行验证，我一般到NCBI()上验证，进去后点击最下面菜单中的B1.AST之三三AA一一三三ht1-E三三三三.三点击这个图标BasicB1.ASTChooseaB1.ASTprogramtorunnuclsoUdsbldstProtioblastblast-SearchanucleotidedatabaseusinganucleotidequeryAlgonthmsblastn,megablast,discontiguousmegaWastSearchproteindatabaseusingaproteinqueryAlgonthms.blastp,ps-blast,ph-Wast.delta-blastSearchproteindatabaseusingatranslatednucleo
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