酶切位点保护碱基

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 sites (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 clos

2、e together in a polylinker), or when selecting enzymes most likely to cleave at the end of a DNA fragment.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, 5 mMDTTand NaCl or KCl depending on the salt requirement of each particular restriction endonucleas

3、e. 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 were 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

4、G109090CCCACATGTGGG129090Asc IGGCGCGCC89090AGGCGCGCCT109090TTGGCGCGCCAA129090Ava ICCCCGGGG85090CCCCCGGGGG109090TCCCCCGGGGGA129090BamH ICGGATCCG81025CGGGATCCCG109090CGCGGATCCGCG129090Bgl IICAGATCTGGAAGATCTTCGGAAGATCTTCC810120752509090BssH IIGGCGCGCC800AGGCGCGCCT1000TTGGCGCGCCAA125090BstE IIGGGT(A/T)ACCC9010BstX IAACTGCAGAACCAATGCATTGG2200AAAACTGCAGCCAATGCATTGGAA242550CTGCAGAACCAATGCATTGGATGCAI 272590Cla ICATCGATG800GATCGATC800CCATCGATGG109090CCCATCGATGGG125050EcoR IGGAATTCC89090CGGAATTCCG109090CC

5、GGAATTCCGG129090Hae IIIGGGGCCCC89090AGCGGCCGCT109090TTGCGGCCGCAA129090Hind IIICAAGCTTG800CCAAGCTTGG1000CCCAAGCTTGGG121075Kpn IGGGTACCC800GGGGTACCCC109090CGGGGTACCCCG129090Mlu IGACGCGTC800CGACGCGTCG102550Nco ICCCATGGG800CATGCCATGGCATG145075Nde ICCATATGG800CCCATATGGG1000CGCCATATGGCG1200GGGIIICAIAIGAAACCC1800GGAATTCCATATGGAATTCC207590GGGAATTCCATATGGAATTCCC227590#欢在下载Nhe IGGCTAGCCCGGCTAGCCGCTAGCTAGCTAG810120101002550Not ITTGCGGCCGCAA1200AIIIGCGGCCGCIIIA161010AAATATGCGGCCGCTATAAA201010ATAAGAATGCGGCCG

6、CTAAACTAT242590AAGGAAAAAAGCGGCCGCAAAAGGAAAA282590Nsi IIGCAIGCAIGCA121090CCAATGCATTGGTTCTGCAGTT229090Pac IHAAHAA800GHAAHAAC10025CCIIAAIIAAGG12090Pme I800GIIIAAACGGIIIAAACC10025GGGIIIAAACCC12050AGCIIIGIIIAAACGGCGCGCCGt；247590Pst IGCIGCAGC800IGCACIGCAGIGCA141010AACIGCAGAACCAAIGCAIIGG229090AAAACTGCAGCCAATGCATTGGAA249090CTGCAGAACCAATGCATTGGATGCAI 2600Pvu ICCGAICGG800AICGAICGAI101025ICGCGAICGCGA12010Sac ICGAGCICG81010Sac IIGCCGCGGC800ICCCCGCGGGGA125090Sal IGICGACGICAAAAGGCCAIAGCGGCCGC!800GCGTCGACGTCTT

7、GGCCATAGCGGCCGCGG1050ACGCGICGACGICGGCCAIAGCGGCCGCG32AA1075Sca IGAGIACIC81025AAAAGIACIIII127575Sma ICCCGGG6010CCCCGGGG8010CCCCCGGGGG101050ICCCCCGGGGGA129090Spe IGACTAGTC81090GGACTAGTCC101090CGGACTAGTCCG12050CTAGACTAGTCTAG14050Sph IGGCATGCC800CATGCATGCATG12025ACATGCATGCATGT141050Stu IAAGGCCTT89090GAAGGCCTTC109090AAAAGGCCIIII129090Xba 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

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