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ID:47403012
大小:110.50 KB
页数:9页
时间:2019-07-04
《PCR保护碱基的设计 连接方法》由会员上传分享,免费在线阅读,更多相关内容在教育资源-天天文库。
1、CleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/µg)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedb
2、ydividingthenumberoftransformantsfromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%."BasePairsfromEnd"referstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthe
3、fragment;thisnumberdoesnotincludethesingle-strandedoverhangfromtheinitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswere
4、roundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesrefertothenumberofindependenttrialsforeachenzymetested(fromMoreira,R.andNoren,C.(1995),Biotechniques,19,56-59).Note:Asageneralrule,enzymesnotlistedbelowrequire6bases
5、pairsoneithersideoftheirrecognitionsitetocleaveefficiently.
6、A
7、B
8、E
9、H
10、K
11、M
12、N
13、P
14、S
15、X
16、 EnzymeBasepairsfromEnd%CleavageEfficiencyVectorInitialCutAatII321 88(2)100(2)95(2) LITMUS29LITMUS28LITMUS29 NcoINcoIPinAI Acc65I21 99(2)75(3) LITMUS29pNEB193 SpeISacI AflII113(2)LIT
17、MUS29StuIAgeI11 100(1)100(2) LITMUS29LITMUS29 XbaIAatII ApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI197(2)LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI21 100(1)8(2) LITMUS39LITMUS38 BsrGIBsrGI BsrGI21 99(2)8
18、8(2) LITMUS39LITMUS38 SphIBspEI BssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI111 100(1)88(1)100(1) LITMUS29LITMUS29LITMUS39 XhoIPstINheI EcoRV1100(2)LITMUS29PstIHindIII321 90(2)91(2)0(2) LITMUS29LITMUS28LITMUS29 NcoINcoIBamHI KasI21 97(1)93(1) LITMUS38L
19、ITMUS38 NgoMIVHindIII KpnI221 100(2)100(2)99(2) LITMUS29LITMUS29pNEB193 SpeISacISacI MluI299(2)LITMUS39EagIMunI2100(1)LITMUS39NgoMIVNcoI2100(1)LITMUS28HindIIINgoMIV21
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