Chapter5IntroductionofDNAintolivingcells-副本课程.pptVIP

Chapter 5 Introduction of DNA into living cells Xinjun Yu 2015.4.14 Contents 5.1 Transformation-the uptake of DNA by bacterial cells 5.2 Identification of recombinants 5.3 Transformation of non-bacterial cells Two purposes of cloning To get a large number of recombinant DNA Purification Ligation production Unligated vector molecules Unligated DNA fragments Self-ligated vector Recombinant DNA carrying the wrong inserted DNA The desired recombinant DNA molecule 5.1 Transformation-the uptake of DNA by bacterial cells Transformation (转化): the introduction of any DNA molecule into any living cell. Competent cell(感受态细胞): A culture of bacteria that has been treated to enhance their ability to take up DNA molecules. Reception cell Prokaryotes : E. coli Bacillus subtilis Pseudomonadaceae Streptococcus Actinomycete Eukaryotes : yeast animal insect 5.1.1 Not all species of bacteria are equally efficient at DNA uptake Bacillus and Streptococcus : not easy to be transformed E.coli : physical and chemical treatment to enhance their ability to take up DNA 5.1.2 Preparation of competent E.coli cells 50mM CaCl2, 4?C : improve DNA binding Heat treatment, 42?C: transport DNA into the cytoplasm 5.1.3 Selection for transformed cells Efficiency of transformation(转化效率): Expressed as transformants per ?g plasmid DNA. 每微克质粒DNA产生的转化子来表示,如106~ 107 转化子/?g DNA Transformation: low efficient (0.01%), Selectable marker is needed Selective medium: to distinguish transformants from non-transformants Larger DNAs transform less efficiency than small DNAs. DNA50kb Fig 5.4 Selecting cells that contain pBR322 plasmids by plating onto agar medium containing ampicillin and/or tetracycline. 对数生长期大肠杆菌50mmol/L CaCl2 , 4oC，30min 加入重组质粒DNA, 冰浴 42oC, 2min. 营养培养基 选择性培养基（含抗生素） Fig 5.5 Ph