稳定酶切位点.doc

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1、CleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/µg)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransf

2、ormantsfromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%."BasePairsfromEnd"referstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedo

3、verhangfromtheinitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Then

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

5、A

6、B

7、E

8、H

9、K

10、M

11、N

12、P

13、S

14、X

15、 EnzymeBasepairsfromEnd%

16、CleavageEfficiencyVectorInitialCutAatII321 88(2)100(2)95(2) LITMUS29LITMUS28LITMUS29 NcoINcoIPinAI Acc65I21 99(2)75(3) LITMUS29pNEB193 SpeISacI AflII113(2)LITMUS29StuIAgeI11 100(1)100(2) LITMUS29LITMUS29 XbaIAatII ApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI

17、197(2)LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI21 100(1)8(2) LITMUS39LITMUS38 BsrGIBsrGI BsrGI21 99(2)88(2) LITMUS39LITMUS38 SphIBspEI BssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI111 100(1)88(1)100(1) LITMUS29LITMUS29LITMUS39 XhoIPstINheI EcoRV1100(

18、2)LITMUS29PstIHindIII321 90(2)91(2)0(2) LITMUS29LITMUS28LITMUS29 NcoINcoIBamHI KasI21 97(1)93(1) LITMUS38LITMUS38 NgoMIVHindIII KpnI221 100(2)100(2)99(2) LITMUS29LITMUS29pNEB193 SpeISacISacI MluI299(2)LITMUS39EagIMunI2100(1)LITMUS39NgoMIVNcoI2100(1)LITMUS28HindIIINgoMIV21