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PCR设计引物时酶切位点的保护碱基表不同内切酶对识别位点以外最少保护碱基数目的要求PCR设计引物时酶切位点的保护碱基表1酶寡核苷酸序列切割率%2hr20hrAccIGGTCGACCCGGTCGACCGCCGGTCGACCGG000000AflIIICACATGTGCCACATGTGGCCCACATGTGGG0909009090AscIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA909090909090AvaICCCCGGGGCCCCCGGGGGTCCCCCGGGGGA509090909090BamHICGGATCCGCGGGATCCCGCGCGGATCCGCG109090259090BglIICAGATCTGGAAGATCTTCGGAAGATCTTCC0752509090BssHIIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA00500090BstEIIGGGT(A/T)ACCC010BstXIAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT0252505090ClaICATCGATGGATCGATCCCATCGATGGCCCATCGATGGG009050009050EcoRIGGAATTCCCGGAATTCCGCCGGAATTCCGG909090909090HaeIIIGGGGCCCCAGCGGCCGCTTTGCGGCCGCAA909090909090HindIIICAAGCTTGCCAAGCTTGGCCCAAGCTTGGG00100075KpnIGGGTACCCGGGGTACCCCCGGGGTACCCCG0909009090MluIGACGCGTCCGACGCGTCG025050NcoICCCATGGGCATGCCATGGCATG050075NdeICCATATGGCCCATATGGGCGCCATATGGCGGGGTTTCATATGAAACCCGGAATTCCATATGGAATTCCGGGAATTCCATATGGAATTCCC0000757500009090NheIGGCTAGCCCGGCTAGCCGCTAGCTAGCTAG0101002550PCR设计引物时酶切位点的保护碱基表2酶寡核苷酸序列切割率%2hr20hrNotITTGCGGCCGCAAATTTGCGGCCGCTTTAAAATATGCGGCCGCTATAAAATAAGAATGCGGCCGCTAAACTATAAGGAAAAAAGCGGCCGCAAAAGGAAAA010102525010109090NsiITGCATGCATGCACCAATGCATTGGTTCTGCAGTT10909090PacITTAATTAAGTTAATTAACCCTTAATTAAGG00002590PmeIGTTTAAACGGTTTAAACCGGGTTTAAACCCAGCTTTGTTTAAACGGCGCGCCGG000750255090PstIGCTGCAGCTGCACTGCAGTGCAAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT0109090001090900PvuICCGATCGGATCGATCGATTCGCGATCGCGA010002510SacICGAGCTCG1010SacIIGCCGCGGCTCCCCGCGGGGA050090SalIGTCGACGTCAAAAGGCCATAGCGGCCGCGCGTCGACGTCTTGGCCATAGCGGCCGCGGACGCGTCGACGTCGGCCATAGCGGCCGCGGAA0101005075ScaIGAGTACTCAAAAGTACTTTT10752575SmaICCCGGGCCCCGGGGCCCCCGGGGGTCCCCCGGGGGA00109010105090SpeIGACTAGTCGGACTAGTCCCGGACTAGTCCGCTAGACTAGTCTAG10100090905050SphIGGCATGCCCATGCATGCATGACATGCATGCATGT001002550StuIAAGGCCTTGAAGGCCTTCAAAAGGCCTTTT909090909090XbaICTCTAGAGGCTCTAGAGCTGCTCTAGAGCACTAGTCTAGACTAG09075750909090XhoICCTCGAGGCCCTCGAGGGCCGCTCGAGCGG0101002575XmaICCCCGGGGCCCCCGGGGGCCCCCCGGGGGGTCCCCCCGGGGGGA02550900759090