primer 60 引物设计教程.docx

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*rc<HKBlHorn*RnoufcllIAZ)HRtocVO»««ncW&0ommyvIW4,2CrsaRKA0>gr*54StrctG«mA6>Fm"Ge<w<ct&MdcmG<n<ne&曲Pe».>7XlJj'*<£王比GefnmeGeOOtfUSebGOPrort«GSSHcwtoGeneMeOGenMeSHMCWWeOSIe>NCBI!“fo<BMMtf09yhto*Mt%*sy*.MdhM*»ro*R*9ccm*Iobcm«-：IMc1110¾yasIRmwt12¾K三OVUOMMPMCPopSetProoeProtenProtonCU5*<sPuQecnPuOCnemCOnPoUrKJPbCnttnrf力/datMmgNCerIGftNC8dMerMtwwe153W<011*<S(*ccU»M«HCWmSU>WWJ10G<ngnb.BBWNC&af4i«5首先选择Nucleotide然后在搜寻栏输入基因序列号（假如不知道基因序列号则输入ratCK18）如图"NCBlISSSEIIWCKldUMQfMC*VX然后点击搜寻，就会出现ratCK18mRNA的基因序列KclWrAXteOtue31.mt>AdcdPMHwEGMOw*nRattusnorvegkuskeratin18(Krt18)1mRNA然因序列号NCBReferenceSe«meMM.0639761FASTAor<<ranKtalr1.OCUSC6F11C11K4*XiS3W1R2KK>三TWW5a11>三三cwJ11affwortl11Wt5InUM1.SMIW3aMUlu>ar>X0MRWFiiwM<v*tkCU9>Mttinl(Xftl9)BKMaM1.M妙”U_2：661>n.aS30?4.1C2：ei»tM7MeKwitwtucv<cuf(IfomaE)¾1t<IMQW>NC¾E<uylBVQ9”Checdit*.Cteru*t.V*ctBE>n*l9KcKma1i*Kut>Aa皿ueh3T£o<”«CHruKodayxttSctu<<n*hl.""4MBn4wdnr*Ktttv.1C5,Iti»nBor4lMF,E1.193rrK.9mmTardl*rcauI.Iemin1/18ter"U"<llKiffr>e>>-e<<c*llultfMUUUrtarpl«7thx0v(haodMinof>h-*adtdac>cy>rkltcnva0FV»S<«1«>.E)tTW1201»百U<M现在，往下拉就会看到基因序列tr.fync*½ms'Itl-ir/ftMvlar(1.rMe=AAB91db.xref=OuSTS21111.S*ORICDIIccatcet<<tctcc<ctcfttdcctctcca(»e&a<*t<a<ceaccecc<cc61CXCtCcttCtcecc*etccc<tccct<ttctctttc<tceee，cw，t121cc<ecctc«MCMeCHcca<cttctatgc<(tgct(<icctcM«rtec“N181MCC贝CtCCCCCUCCtCccctccctCC<t<CCCtccCCKccctCCCCC<M¼CC241t(<cctcca<act(*<C><"M"aa<*cct<*cUE<«CCaC1.cct301M*caa<(tc>><mcc(<aaacc<a<aacs<CM*c1*1a<*Cc*lk*cc<<casta3ltet<cM*MKttcceeMCCCttCMdacttcaceeatc421MNCtggcctc<tcttt<oc*ntctg“,Zm，oCegCSCgtcctgcacat181CBcaat<ccCetG“c”U3tg1ctt7utcctatgMac<caactc<ccat541(c«cca(t1.ct<<*<ct(MattE“c1cc(cm<Ct<CtCC*t<cc<ucat601cae<a<(t<ca<c1<<*<*c*cHc,a<cgctea(«v«ct(ct(ttca1*a661(aa<*aefetCM4MC*Mtee*Mtcctwctca<tt*CCNttctc<Cttc721”««Wlg<atfCtCCCMRCtCMeEwClCNyCKICfttCCftK?81CClCtat3HCCtCMWXcf"MgactfWMCtdetCetUC“ca841(«1，CaMaCMcaccc,a<tcaccacCM<tct(CC(»Mca(c，cd“x901ce*cctt<«“U«“ec>cEcc<acct(<*<st(acct<(aeteaca961a*a<eca4*eaCMCc<aocwctctCC*MtCcM<ca<tcacyc1021，hcmctc*ktct(<tccttct<catct<ca*tcftEyCcCtyIOSiM&KCCKccc<ccocMCaMUCaMCCctet”axH8g4tcacr<aIIQImat，cccctacc«c“ct，BetCC*CCCCC«x«atttcCtctCa1201c<cccc<*ctcca<ca*cccstc>*ct<tcca<>CK*e*c*ccc1261，<M，,c“aCt(<tttce<a<cd,accMMttCtydwca<1321RWaXcccttgc(ftytecRttyweezam2aIWlaawaaa"«a&»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, UPgPdeAvailableAnVfigradraVnihh)Ponthesp11t.DoyouWaIUtodownloaditnow?UPgIadeDeSerdon:UpgradeInChJdeSthefbDowinfeatures Supponforthelatestgcno11ucBabaSeSavailableattheNCBlB1.ASTWrVer SupportforWindows8Forplease5Xhtto"wwwOrElKTbo某OftCorn/ProductgZdEyvVpgradgghtmlKtoctnfornabonISaadabCeat1即/TwwwF<rengfqogofcnCallorsupportUneM65O562703WandngOwmgtothechangemtheJavaVefsJgported,theSmartUpdaterWlnnotworkPleasedownloadandCistailPnmPrnjcr620fromourwebcchlto"wwwEaroertimoRCgVCTTM3P'co11VpbcE¾clGtWdc>PocUjgtgn破解版打开会出现这个对话框，关闭即可FileEditViewAnalyzeToolsOnline点击新建将序列更制至这个对话框NewSequenceSequenceDMnitionJNucleotidesequenceSequenceGCA9611021AAACCAGAJICGATGGAGcAATCCTTGGCtcaatgggcAGCACCTCGGGGMGTGGAGCCCAGATCAGG-jAAACTTCCTTCTGCATctggaatcaGAGCTGGCacGGC1081AGAAGGACAGCGeCAGACCCASAATACGAAgccctcttgAACATeAAGGTCAAOTGA1141GGcGGAGATTGccacctaccGccgcttgctGGGGTGGGGACGATTTCAGTCTC.CGAIZOlCGCCCTGGACTccagcaactCcatgcaaacTgtccagaggACAACTACCCGTAAG-CGT1261GGATGGCAGtgotgtccgAGACCAATGATACCAGAGTTCTGAGGCACTAGGC,AGA1321AGj1.AGGGAJlCCCCTTGGGGACTGJ1.GGGACCMTaJ1.AAGTTTGTCCJUIMAJ1.AA1381kkkkkkAAAAAAAVJMAddICancelIHelp点击AddFoe£d<MICWM>田TooK0rtnHelpA0/x”|0,51|也匍鼻=Wl£-Se<uehceInformaflonSearchStatus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