酶切位点保护碱基

Toaccessyouraccount,loginorregister.shoppingcart|loginRequestTechnicalSupportEnzymeFinderNEBcutterNEBufferChartDoubleDigestFinderIsoschizomersDNASequencesandMapsREBASEHomeTechnicalReferenceRestrictionEndonucleasesCleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/µg)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransformantsfromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%.BasePairsfromEndreferstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedoverhangfromtheinitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesrefertothenumberofindependenttrialsforeachenzymetested(fromMoreira,R.andNoren,C.(1995),Biotechniques,19,56-59).Note:Asageneralrule,enzymesnotlistedbelowrequire6basespairsoneithersideoftheirrecognitionsitetocleaveefficiently.|A|B|E|H|K|M|N|P|S|X|EnzymeBasepairsfromEnd%CleavageEfficiencyVectorInitialCutAatII32188(2)100(2)95(2)LITMUS29LITMUS28LITMUS29NcoINcoIPinAIAcc65I2199(2)75(3)LITMUS29pNEB193SpeISacIAflII113(2)LITMUS29StuIAgeI11100(1)100(2)LITMUS29LITMUS29XbaIAatIIApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI197(2)LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI21100(1)8(2)LITMUS39LITMUS38BsrGIBsrGIBsrGI2199(2)88(2)LITMUS39LITMUS38SphIBspEIBssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI111100(1)88(1)100(1)LITMUS29LITMUS29LITMUS39XhoIPstINheIEcoRV1100(2)LITMUS29PstIHindIII32190(2)91(2)0(2)LITMUS29LITMUS28LITMUS29NcoINcoIBamHIKasI2197(1)93(1)LITMUS38LITMUS38NgoMIVHindIIIKpnI221100(2)100(2)99(2)LITMUS29LITMUS29pNEB193SpeISacISacIMluI299(2)LITMUS39EagIMunI2100(1)LITMUS39NgoMIVNcoI2100(1)LITMUS28HindIIINgoMIV2100(1)LITMUS39MunINheI12100(1)82(1)LITMUS39LITMUS39EcoRIEagINotI741100(2)100(1)98(2)BluescriptSK-BluescriptSK-BluescriptSK-SpeIKspIXbaINsiI332100(2)77(4)95(2)LITMUS29LITMUS29LITMUS28BssHIIBglIIBssHIIPacI176(3)pNEB193BamHIPmeI194(2)pNEB193PstIPstI32198(1)50(5)37(3)LITMUS29LITMUS39LITMUS29EcoRVHindIIIEcoRISacI199(2)LITMUS29AvrIISalI32189(2)23(2)61(3)LITMUS39LITMUS39LITMUS38SpeISphISphISfiI94181(2)97(2)93(2)LITMUS38LITMUS38LITMUS38BamHIMluIEcoRISpeI22100(2)100(2)LITMUS29LITMUS29Acc65IKpnISphI22199(1)97(1)92(2)LITMUS39LITMUS39LITMUS38SalIBsrGISalIXbaI1199(2)94(1)LITMUS29LITMUS29AgeIPinAIXhoI197(2)LITMUS29EcoRIXmaI2298(1)92(1)pNEB193pNEB193AscIBssHIIToaccessyouraccount,loginorregister.shoppingcart|loginRequestTechnicalSupportEnzymeFinderNEBcutterNEBufferChartDoubleDigestFinderIsoschizomersDNASequencesandMapsREBASEHomeTechnicalReferenceRestrictionEndonucleasesCleavageClosetotheEndofDNAFragments(oligonucleotides)Totestthevaryingrequirementsrestrictionendonucleaseshaveforthenumberofbasesflankingtheirrecognitionsequences,aseriesofshort,double-strandedoligonucleotidesthatcontaintherestrictionendonucleaserecognitionsites(showninred)weredigested.Thisinformationmaybehelpfulwhenchoosingtheorderofadditionoftworestrictionendonucleasesforadoubledigest(aparticularconcernwhencleavingsitesclosetogetherinapolylinker),orwhenselectingenzymesmostlikelytocleaveattheendofaDNAfragment.Theexperimentwasperformedasfollows:0.1A260unitofoligonucleotidewasphosphorylatedusingT4polynucleotidekinaseandγ-[32P]ATP.1µgof5´[32P]-labeledoligonucleotidewasincubatedat20°Cwith20unitsofrestrictionendonucleaseinabuffercontaining70mMTris-HCl(pH7.6),10mMMgCl2,5mMDTTandNaClorKCldependingonthesaltrequirementofeachparticularrestrictionendonuclease.Aliquotsweretakenat2hoursand20hoursandanalyzedby20%PAGE(7Murea).Percentcleavagewasdeterminedbyvisualestimateofautoradiographs.Asacontrol,self-ligatedoligonucleotideswerecleavedefficiently.Decreasedcleavageefficiencyforsomeofthelongerpalindromicoligonucleotidesmaybecausedbytheformationofhairpinloops.|A|B|C|E|H|K|M|N|P|S|X|EnzymeOligoSequenceChainLength%Cleavage2hr20hrAccIGGTCGACCCGGTCGACCGCCGGTCGACCGG81012000000AflIIICACATGTGCCACATGTGGCCCACATGTGGG810120909009090AscIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA81012909090909090AvaICCCCGGGGCCCCCGGGGGTCCCCCGGGGGA81012509090909090BamHICGGATCCGCGGGATCCCGCGCGGATCCGCG81012109090259090BglIICAGATCTGGAAGATCTTCGGAAGATCTTCC810120752509090BssHIIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA8101200500090BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT2224270252505090ClaICATCGATGGATCGATCCCATCGATGGCCCATCGATGGG881012009050009050EcoRIGGAATTCCCGGAATTCCGCCGGAATTCCGG81012909090909090HaeIIIGGGGCCCCAGCGGCCGCTTTGCGGCCGCAA81012909090909090HindIIICAAGCTTGCCAAGCTTGGCCCAAGCTTGGG8101200100075KpnIGGGTAC