• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Cleavage Efficiency Close to the&nbs

        互联网

        1259
         

        When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE, it is very important to determine how many extra nucleotides should be added to the 5’-end of the PCR primer next to the introduced recognition site to ensure efficient cleavage with the appropriate restriction endonuclease. Some restriction endonucleases cleave DNA poorly when their recognition sites are located at the ends of DNA fragments. Information on restriction endonuclease performance close to the end of DNA fragments may be helpful when planning the introduction of cleavage sites at DNA termini in PCR experiments.

        Experiments were performed as follows: PCR primers having 1-5 extra nucleotides at the 5’-end of the PCR primers adjacent to the introduced recognition site were 5’-end labeled with [32 P] by T4 Polynucleotide Kinase and used in the PCR reaction. Purification of PCR fragment was performed using the modified glass beads procedure as indicated in the DNA Extraction Kit (#K0513) followed by ethanol precipitation. DNA (0.5µg) and 10u of restriction endonuclease were incubated for 1 hour at the recommended incubation temperature and optimal buffer for each enzyme in a total volume of 40µl. Reaction products were separated by 10% PAGE and percent cleavage was determined using OptiQuant Image Analysis Software.

          Cleavage efficiency:
          - 0%
          - 0-20%
          - 20-50%
          - 50-100%
        - incubation was performed for 16 hours
        nd - not determined
         
        Enzyme Base pairs from site to fragment end
        1 2 3 4 5
        AarI  20-50  50-100
        AatII  0 0-20 20-50  50-100
        Acc65I  0 20-50
        AdeI  50-100
        AluI 0  20-50
        Alw21I  50-100
        Alw26I  50-100
        Alw44I  0 20-50 50-100
        BamHI  50-100
        BcnI  20-50 50-100
        BclI  0 20-50 nd
        BcuI  50-100
        BfiI  50-100
        BfmI  50-100
        BglI  20-50 50-100
        BglII  0 50-100
        Bme1390I  20-50 50-100
        BpiI  50-100
        Bpu10I  20-50 50-100
        Bpu1102I  50-100
        BseDI  0 50-100
        BseGI  50-100
        BseLI  0 50-100
        BseMI  0-20 50-100
        BseMII  50-100
        BseNI  0 50-100
        BseSI  0 0-20 nd
        BseXI  20-50 50-100
        Bsh1236I  50-100
        Bsh1285I  0-20 50-100
        BshNI  50-100
        BshTI  20-50 50-100
        Bsp68I  0 50-100
        Bsp119I  50-100
        Bsp120I  20-50 50-100
        Bsp143I  50-100
        Bsp143II  0 50-100
        Bsp1407I  20-50 50-100
        BspLI  50-100
        BspPI  0 50-100
        BspTI  0 0-20 50-100
        Bst1107I  0-20 50-100
        BstXI  0 50-100
        Bsu15I  50-100
        BsuRI  0-20 20-50 50-100
        Cleavage Efficiency Close to the&nbs
        Enzyme Base pairs from site to fragment end
        1 2 3 4 5
        CaiI  0 0-20
        CfrI  0 50-100
        Cfr9I  20-50 50-100
        Cfr10I  20-50 50-100
        Cfr13I  50-100
        Cfr42I  50-100
        CpoI  50-100
        Csp6I  50-100
        DraI  0 0-20 50-100
        Eam1104I  0 50-100
        Eam1105I  0 50-100
        Ecl136II  50-100
        Eco24I  50-100
        Eco31I  20-50 50-100
        Eco32I  20-50 50-100
        Eco47I  50-100
        Eco47III  0 0-20 50-100
        Eco52I   0-20  50-100
        Eco57I  50-100
        Eco57MI  50-100
        Eco72I  0-20 50-100
        Eco81I  50-100
        Eco88I  50-100
        Eco91I  20-50 50-100
        Eco105I  20-50 50-100
        Eco130I  0 50-100
        Eco147I  0 50-100
        EcoO109I  50-100
        EcoRI  50-100
        EheI  20-50 50-100
        Esp3I  50-100
        Cleavage Efficiency Close to the&nbs
        Enzyme Base pairs from site to fragment end
        1 2 3 4 5
        FspAI  0-20 20-50 50-100
        GsuI  50-100
        Hin1I  50-100
        Hin6I  0 0-20 50-100
        HincII  50-100
        HindIII  0 0-20 50-100
        HinfI  50-100
        HpaII 0-20  50-100
        HphI  0 50-100
        Hpy8I  0-20 50-100
        KpnI  0 20-50
        Kpn2I  0 50-100
        KspAI  20-50 50-100
        LweI  20-50 50-100
        MbiI  0 0-20 50-100
        MboI  50-100
        MboII  0 20-50 nd
        MlsI  0-20 50-100
        MluI  20-50 50-100
        MnlI  0 50-100
        Mph1103I  0-20 nd
        MspI 0-20  50-100
        MssI  20-50 50-100
        MunI  20-50 50-100
        MvaI 20-50 50-100
        Mva1269I  0 50-100
        NcoI  0 50-100
        NdeI  0-20 20-50
        NheI  0 20-50 50-100
        NmuCI  20-50 50-100
        NotI  20-50 50-100
        NsbI  0 0-20 50-100
        Cleavage Efficiency Close to the&nbs
        Enzyme Base pairs from site to fragment end
        1 2 3 4 5
        OliI  0-20 20-50 50-100
        PaeI  0 0-20 20-50 50-100
        PagI  20-50 50-100
        PauI  0 50-100
        PdiI  0-20 50-100
        PdmI  0-20 20-50 nd
        Pfl23II  0 20-50 nd
        Psp5II  0 50-100
        Psp1406I  0-20 50-100
        PstI 0-20  50-100
        PsuI  0-20 20-50 nd
        PsyI  0 50-100
        PvuI  20-50 50-100
        PvuII  50-100
        SacI  50-100
        SalI  20-50 50-100
        SatI  0 50-100
        ScaI  0-20 50-100
        SchI  50-100
        SdaI  0 0-20 20-50
        SduI  50-100
        SmaI  50-100
        SmiI  0-20 50-100
        SspI  0-20 50-100
        TaaI  20-50 50-100
        TaiI  50-100
        TaqI 0  20-50 50-100
        TasI 0  20-50 50-100
        TatI  0-20 20-50 50-100
        TauI  20-50 50-100
        Tru1I  0 0-20 50-100
        Van91I 0  50-100
        VspI  50-100
        XagI  20-50 50-100
        XapI  20-50 50-100
        XbaI  20-50 50-100
        XceI  0 20-50 nd
        XhoI 0-20  50-100
        XmaJI  50-100
        XmiI  50-100
         
        <center> <p>  </p> </center>
        上一篇:Blunt end cloning of PCR produc   下一篇:Purification of PCR products
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序