2024年3月10日发(作者：干谷菱)
靠近DNA片段末端识别位点的酶切效率
当识别位点靠近DNA分子末端时，有些限制酶的酶切效率非常低。表列出了Fermentas公司限制酶的识别位点靠近PCR片段末端不同碱基数的切
割效率。 实验设计如下：
PCR引物设计时在限制酶识别序列5'-端增加1-5个碱基。引物的5'-端经T4多聚核苷激酶 (#EK0031)标记[P]基团。采用该引物扩增的PCR产
物可经Silica Bead DNA Gel Extraction Kit (#K0513)纯化和乙醇沉淀。纯化的PCR产物(0.5 μg)与10 units限制酶在优化缓冲液和优化反
应温度下孵育1小时。OptiQuant®成像软件分析酶切产物的电泳结果(10% PAGE凝胶)，判断酶切效率。 酶切时间为16小时。
表. 靠近PCR片段末端识别位点的酶切效率
Bp from the recognition site to fragment end
Enzyme
1
AanI (PsiI)
AarI
32
2
0-20
3
20-50
50-100
4 5
0
20-50
AasI (DrdI)
AatII
50-100
0 0-20
50-100
20-50 50-100
Acc65I (Asp718I)
AdeI (DraIII)
AjiI (BmgBI)
AluI
0-20
50-100
50-100
0-20 20-50 50-100
Alw21I (BsiHKAI)
Alw26I
Alw44I
ApaI
50-100
50-100
0
50-100
20-50 50-100
BamHI50-100
BauI (BssSI)
BclI
0-20
0
20-50
50-100
50-100
BcnI (NciI)
BcuI (SpeI)
BfiI (BmrI)
BfmI (SfcI)
20-50
50-100
50-100
50-100
50-100
BfuI (BciVI)
BglI
50-100
20-50 50-100
50-100
50-100
50-100
BglII0
Bme1390I (ScrFI)
BoxI (PshAI)
BpiI (BbsI)
Bpu10I
20-50
0
50-100
20-50 50-100
Bpu1102I (BlpI)
BseDI (BsaJI)
BseGI (BtsCI)
BseJI (BsaBI)
BseLI (BslI)
50-100
0
50-100
0
0
50-100
50-100
50-100
50-100
BseMI (BsrDI)0-20
BseMII (BspCNI)
BseNI (BsrI)
50-100
0 50-100
BseSI (Bme1580I)
BseXI (BbvI)
50-100
20-50 50-100
Bsh1236I (BstUI)
Bsh1285I (BsiEI)
BshNI (BanI)
BshTI (AgeI)
50-100
0-20
50-100
20-50 50-100
50-100
Bsp119I (BstBI)50-100
Bsp120I (PspOMI)
Bsp1407I (BsrGI)
20-50
20-50
50-100
0
50-100
0
0
50-100
50-100
Bsp143I (Sau3AI)
Bsp68I (NruI)
BspLI (NlaIV)
BspOI (BmtI)
BspPI (AlwI)
50-100
20-50
50-100
0-20
50-100
50-100
BspTI (AflII)0 50-100
Bst1107I (BstZ17I)0-20
BstXI0 50-100
Bsu15I (ClaI)50-100
BsuRI (HaeIII)
BveI (BspMI)
CaiI (AlwNI)
0-20
0-20
0
20-50 50-100
50-100
0-20
50-100
50-100
Cfr10I (BsrFI)20-50
Cfr13I (Sau96I)
Cfr42I (SacII)
Cfr9I (XmaI)
CfrI (EaeI)
50-100
50-100
20-50
0
50-100
50-100
CpoI (RsrII)
CseI (HgaI)
50-100
50-100
Csp6I (CviQI)
DpnI
DraI
50-100
50-100
0 0-20
50-100
50-100
50-100
Eam1104I (EarI)
Eam1105I (AhdI)
0
0
Ecl136II (EcoICRI)50-100
Eco105I (SnaBI)
Eco130I (StyI)
Eco147I (StuI)
Eco24I (BanII)
Eco31I (BsaI)
20-50
0
0
50-100
20-50
50-100
50-100
50-100
50-100
50-100 Eco32I (EcoRV)
Eco47I (AvaII)
20-50
50-100
Eco47III (AfeI)
Eco52I (EagI)
0
0-20
50-100
50-100
0-20
50-100
50-100
Eco57I (AcuI)
Eco57MI
2024年3月10日发(作者：干谷菱)
靠近DNA片段末端识别位点的酶切效率
当识别位点靠近DNA分子末端时，有些限制酶的酶切效率非常低。表列出了Fermentas公司限制酶的识别位点靠近PCR片段末端不同碱基数的切
割效率。 实验设计如下：
PCR引物设计时在限制酶识别序列5'-端增加1-5个碱基。引物的5'-端经T4多聚核苷激酶 (#EK0031)标记[P]基团。采用该引物扩增的PCR产
物可经Silica Bead DNA Gel Extraction Kit (#K0513)纯化和乙醇沉淀。纯化的PCR产物(0.5 μg)与10 units限制酶在优化缓冲液和优化反
应温度下孵育1小时。OptiQuant®成像软件分析酶切产物的电泳结果(10% PAGE凝胶)，判断酶切效率。 酶切时间为16小时。
表. 靠近PCR片段末端识别位点的酶切效率
Bp from the recognition site to fragment end
Enzyme
1
AanI (PsiI)
AarI
32
2
0-20
3
20-50
50-100
4 5
0
20-50
AasI (DrdI)
AatII
50-100
0 0-20
50-100
20-50 50-100
Acc65I (Asp718I)
AdeI (DraIII)
AjiI (BmgBI)
AluI
0-20
50-100
50-100
0-20 20-50 50-100
Alw21I (BsiHKAI)
Alw26I
Alw44I
ApaI
50-100
50-100
0
50-100
20-50 50-100
BamHI50-100
BauI (BssSI)
BclI
0-20
0
20-50
50-100
50-100
BcnI (NciI)
BcuI (SpeI)
BfiI (BmrI)
BfmI (SfcI)
20-50
50-100
50-100
50-100
50-100
BfuI (BciVI)
BglI
50-100
20-50 50-100
50-100
50-100
50-100
BglII0
Bme1390I (ScrFI)
BoxI (PshAI)
BpiI (BbsI)
Bpu10I
20-50
0
50-100
20-50 50-100
Bpu1102I (BlpI)
BseDI (BsaJI)
BseGI (BtsCI)
BseJI (BsaBI)
BseLI (BslI)
50-100
0
50-100
0
0
50-100
50-100
50-100
50-100
BseMI (BsrDI)0-20
BseMII (BspCNI)
BseNI (BsrI)
50-100
0 50-100
BseSI (Bme1580I)
BseXI (BbvI)
50-100
20-50 50-100
Bsh1236I (BstUI)
Bsh1285I (BsiEI)
BshNI (BanI)
BshTI (AgeI)
50-100
0-20
50-100
20-50 50-100
50-100
Bsp119I (BstBI)50-100
Bsp120I (PspOMI)
Bsp1407I (BsrGI)
20-50
20-50
50-100
0
50-100
0
0
50-100
50-100
Bsp143I (Sau3AI)
Bsp68I (NruI)
BspLI (NlaIV)
BspOI (BmtI)
BspPI (AlwI)
50-100
20-50
50-100
0-20
50-100
50-100
BspTI (AflII)0 50-100
Bst1107I (BstZ17I)0-20
BstXI0 50-100
Bsu15I (ClaI)50-100
BsuRI (HaeIII)
BveI (BspMI)
CaiI (AlwNI)
0-20
0-20
0
20-50 50-100
50-100
0-20
50-100
50-100
Cfr10I (BsrFI)20-50
Cfr13I (Sau96I)
Cfr42I (SacII)
Cfr9I (XmaI)
CfrI (EaeI)
50-100
50-100
20-50
0
50-100
50-100
CpoI (RsrII)
CseI (HgaI)
50-100
50-100
Csp6I (CviQI)
DpnI
DraI
50-100
50-100
0 0-20
50-100
50-100
50-100
Eam1104I (EarI)
Eam1105I (AhdI)
0
0
Ecl136II (EcoICRI)50-100
Eco105I (SnaBI)
Eco130I (StyI)
Eco147I (StuI)
Eco24I (BanII)
Eco31I (BsaI)
20-50
0
0
50-100
20-50
50-100
50-100
50-100
50-100
50-100 Eco32I (EcoRV)
Eco47I (AvaII)
20-50
50-100
Eco47III (AfeI)
Eco52I (EagI)
0
0-20
50-100
50-100
0-20
50-100
50-100
Eco57I (AcuI)
Eco57MI