pcr保护碱基的设计连接方法

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1、CleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/pg)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransformants

2、fromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%.••BasePairsfromEnd”referstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedoverhangfromth

3、einitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesr

4、efertothenumberofindependenttrialsforeachenzymetested(fromMoreira，R.andNoren,C.(1995)，Biotechniques,19,56-59).Note:Asageneralrule，enzymesnotlistedbelowrequire6basespairsoneithersideoftheirrecognitionsitetocleaveefficiently.A

5、B

6、E

7、H

8、K

9、M

10、N

11、P

12、S

13、X

14、EnzymeBasepairsfromEnd%CleavageEfficiencyVectorIniti

15、alCutAatll388(2)LITMUS29NeoI2100(2)LITMUS28NeoI195(2)LITMUS29PinAIAcc65I299(2)LITMUS29SpeI175(3)pNEB193SacIAflII113(2)LITMUS29StuIAgeI1100(1)LITMUS29Xbal1100(2)LITMUS29AatllApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvril1100(2)LITMUS29SacIBamHI197(2)LITMUS29HindIIIBglll3100(2)LITMUS29NsiIBsi

16、WI2100(2)LITMUS29BssHIIBspEI2100(1)LITMUS39BsrGI18(2)LITMUS38BsrGIBsrG1299(2)LITMUS39SphI188(2)LITMUS38BspEIBssHII2100(2)LITMUS29BsiWIEag12100(2)LITMUS39NheIEcoR11100(1)LITMUS29XhoI188⑴LITMUS29Pstl1100(1)LITMUS39NheIEcoRV1100(2)LITMUS29PstlHindIII390(2)LITMUS29NeoI291(2)LITMUS28NeoI10(2)LITMUS2

17、9BamHIKas1297⑴LITMUS38NgoMIV193⑴LITMUS38HindIIIKpn12100(2)LITMUS29SpeI2100(2)LITMUS29SacI199(2)pNEB193SacIMlu1299(2)LITMUS39EagIMun12100(1)LITMUS39NgoMIVNeo12100(1)LITMUS28HindIIINgoMIV2100(1)LITMUS39MunINhe11100(1)LITMUS39EcoRI28