基因克隆分析课件

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1、基因的克隆、转化与表达DNA重组技术（DNArecombinanttechniques）基因克隆（GeneCloning）工具酶限制型内切酶（RestrictionEnzymes）磷酸酶（phosphatase）激酶（Kinase）连接酶（Ligase）目的片断载体限制-修饰体系——由限制性内切核酸酶与甲基化酶组成限制性内切核酸酶EcoRI5’-G↓AATTC-3’GAATTC3’-CTTAA↑G-5’CTTAA–5’GBamHIGGATCC5’端粘性末端CCTAGGPstI5’-CTGCA↓G-3’3’端粘性末端3’-G↑ACGTC-5’HpaI5’-GTT↓AAC-3’平末端3’-

2、CAA↑TTG-5’相配末端（CompatibleEnds）同一种内切酶平末端EcoRV和HincIIBamHI和BglIIGGATCCAGATCTCCTAGGTCTAGASalI和XhoIGTCGACCTCGAGCAGCTGGAGCTCStaractivity:在极端非标准条件下，限制性内切酶可以识别并切开与其特定序列相似但并不一样的序列。这种改变了特异性的特性被称为星活性。BamHIBsiWIBsoBIDpnIIEcoRIHgaIPflMIPvuIISalIScaISspITth111IHighglycerolconcentration[>5%v/v]Highunitstoµgof

3、DNAratio[Varieswitheachenzyme,usually>100units/µg]Lowionicstrength[<25mM]HighpH[>pH8.0]Presenceoforganicsolvents[DMSO,ethanol(5),ethyleneglycol,dimethylacetamide,dimethylformamide,sulphalane(10)]SubstitutionofMg++withotherdivalentcations[Mn++,Cu++,Co++,Zn++]酶切反应体系DNA0.1ug/ul10XBuffer1XDI－H2OEnzy

4、me3-5unit/ugDNABuffer的选择SuggestedNEBuffersforDoubleDigestionEnzymeAatIIAvrIIBamHIBglIIBsgIEagIEcoRIEcoRVHindIIIKpnIMseINcoINdeINheINotIPstIPvuISacISacIISalISmaISpeISphIXbaIXhoIXmaINEBuffer423343EcoRI3212342333143422224AatII4--4seqseq4seqseq4444444seq4seq44seq444444AvrII24--3243EcoRI2212242332143

5、422224BamHI3seq3--333EcoRI3seqseq333seq333seqseq3seqseq333seqBglII3seq23--33EcoRI32223323332seq3seq2223seqBsgI44433--seqseq42seq2444333443424244EagI3seq333seq--EcoRI3seqseq333seq333seqseq3seqseq333seqEcoRIEcoRIseqEcoRIEcoRIEcoRIseqEcoRI--EcoRIEcoRI1EcoRIEcoRIEcoRIEcoRIEcoRIEcoRIEcoRI1EcoRIEcoRIs

6、eqEcoRIEcoRIseqEcoRIseqEcoRV3423343EcoRI--222322333223422234HindIII242seq22seqEcoRI2--2222222222seq42222seqKpnI141seq2seqseq122--111121214seqseq11214MseI2423223EcoRI221--222233223422224NcoI3423343EcoRI3212--42333143422224NdeI4443343EcoRI22124--4333443422244NheI242seq24seqEcoRI221224--22214seq422

7、224NotI3seq33333EcoRI3222332--33223seq22332PstI3433333EcoRI32133323--3123422334PvuI3seq23333EcoRI322333233--223seq2233seqSacI141seq24seq12212141212--4seq411414SacII444seqseq4seqEcoRI22424442224--seq424244SalI3seq33333EcoRI3s