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1、精品文档Cleavage Close to the End of DNA Fragments (oligonucleotides)To test the varying requirements restriction endonucleases have for the number of bases flanking their recognition sequences, a series of short, double-stranded oligonucleotides that contain the restriction endonuclease recognition sit
2、es (shown in red) were digested. This information may be helpful when choosing the order of addition of two restriction endonucleases for a double digest (a particular concern when cleaving sites close together in a polylinker), or when selecting enzymes most likely to cleave at the end of a DNA fra
3、gment.The experiment was performed as follows: 0.1 A 260 unit of oligonucleotide was phosphorylated using T4 polynucleotide kinase and- 32P ATP. 1 闻of 5 32P-labeled oligonucleotide was incubated at 20C with 20 unitsof restriction endonuclease in a buffer containing 70 mM Tris-HCl (pH7.6), 10 mMMgC2,
4、 5 mMDTTand NaCl or KCl depending on the salt requirement of each particular restriction endonuclease. Aliquots were taken at 2hours and 20 hours and analyzed by 20% PAGE (7 M urea). Percent cleavage was determined by visual estimate of autoradiographs.As a control, self-ligated oligonucleotides wer
5、e cleaved efficiently. Decreased cleavage efficiency for some of the longer palindromic oligonucleotides may be caused by the formation of hairpin loops.|A|B|C|E|H|K|M|N |P|S|X|EnzymeOligo SequenceChainLength% Cleavage2 hr20 hrAcc IGGTCGACC800CGGTCGACCG1000CCGGTCGACCGG1200Afl IIICACATGTG800CCACATGTG
6、G109090CCCACATGTGGG129090Asc IGGCGCGCC89090AGGCGCGCCT109090TTGGCGCGCCAA129090Ava ICCCCGGGG85090CCCCCGGGGG109090TCCCCCGGGGGA129090BamH ICGGATCCG81025CGGGATCCCG109090CGCGGATCCGCG129090Bgl IICAGATCTGGAAGATCTTCGGAAGATCTTCC810120752509090BssH IIGGCGCGCC800AGGCGCGCCT1000TTGGCGCGCCAA125090BstE IIGGGT(A/T)A
7、CCC9010BstX IAACTGCAGAACCAATGCATTGG2200AAAACTGCAGCCAATGCATTGGAA242550CTGCAGAACCAATGCATTGGATGCAI 272590Cla ICATCGATG800GATCGATC800CCATCGATGG109090CCCATCGATGGG125050EcoR IGGAATTCC89090CGGAATTCCG109090CCGGAATTCCGG129090Hae IIIGGGGCCCC89090AGCGGCCGCT109090TTGCGGCCGCAA129090Hind IIICAAGCTTG800CCAAGCTTGG1
8、000CCCAAGCTTGGG121075Kpn IGGGTACCC800GGGGTACCCC109090CGGGGTACCCCG129090Mlu IGACGCGTC800CGACGCGTCG102550Nco ICCCATGGG800CATGCCATGGCATG145075Nde ICCATATGG800CCCATATGGG1000CGCCATATGGCG1200GGGIIICAIAIGAAACCC1800GGAATTCCATATGGAATTCC207590GGGAATTCCATATGGAATTCCC227590#欢在下载Nhe IGGCTAGCCCGGCTAGCCGCTAGCTAGCTA
9、G810120101002550Not ITTGCGGCCGCAA1200AIIIGCGGCCGCIIIA161010AAATATGCGGCCGCTATAAA201010ATAAGAATGCGGCCGCTAAACTAT242590AAGGAAAAAAGCGGCCGCAAAAGGAAAA282590Nsi IIGCAIGCAIGCA121090CCAATGCATTGGTTCTGCAGTT229090Pac IHAAHAA800GHAAHAAC10025CCIIAAIIAAGG12090Pme I800GIIIAAACGGIIIAAACC10025GGGIIIAAACCC12050AGCIIIGI
10、IIAAACGGCGCGCCGt；247590Pst IGCIGCAGC800IGCACIGCAGIGCA141010AACIGCAGAACCAAIGCAIIGG229090AAAACTGCAGCCAATGCATTGGAA249090CTGCAGAACCAATGCATTGGATGCAI 2600Pvu ICCGAICGG800AICGAICGAI101025ICGCGAICGCGA12010Sac ICGAGCICG81010Sac IIGCCGCGGC800ICCCCGCGGGGA125090Sal IGICGACGICAAAAGGCCAIAGCGGCCGC!800GCGTCGACGTCTT
11、GGCCATAGCGGCCGCGG1050ACGCGICGACGICGGCCAIAGCGGCCGCG32AA1075Sca IGAGIACIC81025AAAAGIACIIII127575Sma ICCCGGG6010CCCCGGGG8010CCCCCGGGGG101050ICCCCCGGGGGA129090Spe IGACTAGTC81090GGACTAGTCC101090CGGACTAGTCCG12050CTAGACTAGTCTAG14050Sph IGGCATGCC800CATGCATGCATG12025ACATGCATGCATGT141050Stu IAAGGCCTT89090GAAG
12、GCCTTC109090AAAAGGCCIIII129090Xba ICTCTAGAG800GCTCTAGAGC109090TGCTCTAGAGCA127590CTAGTCTAGACTAG147590Xho ICCTCGAGG800CCCTCGAGGG101025CCGCTCGAGCGG121075Xma ICCCCGGGG800CCCCCGGGGG102575CCCCCCGGGGGG125090TCCCCCCGGGGGGA149090Cleavage Close to the End of DNA Fragments (linearized vector)Linearized vectors were incubated with the indicated enzymes (10 units/闻)for 60minutes at the recommended incubation temperature and NEBuffer for each enzyme.Following ligation and transformation, c
