Chapter3PurificationofDNAfromLivingCells副本课程.ppt

Three kinds of DNA for genetic engineering Plasmid DNA Genome DNA Phage DNA Chapter 3 Purification of DNA from Living Cells Xinjun Yu, 于欣君 2015.3.24 Contents 3.1 Preparation of total cell DNA 3.1.1 Growing and harvesting a bacterial culture 3.1.2 Preparation of a cell extract 3.1.3 Purification of DNA from a cell extract 3.1.4 Concentration of DNA samples 3.1.5 Measurement of DNA concentration 3.1.6 Preparation of total cell DNA from organisms other than bacteria 3.2 Preparation of plasmid DNA 3.2.1 Separation on the basis of size 3.2.2 Separation on the basis of conformation Alkaline denaturation Ethidium bromide-caesium chloride density gradient centrifugation Plasmid amplification. 3.1 Preparation of total cell DNA Four steps for total DNA preparation from bacteria: Cell culture and harvested Cell broken/ cell extract DNA purify DNA concentrated. 3.1.1 Growing and harvesting a bacterial culture Broth culture: 肉汤培养基 Example of a defined medium: M9 Undefined medium: LB (Luria-Bertani) 3.1.1 Growing and harvesting a bacterial culture Bacterial culture as a source of DNA: LB medium LB medium, 37?C, 150-250 rpm, to maximum density of 2-3*109 cells/ml For E.coli, OD600=1, ~ 0.8x109 cells/ml 3.1.2 Preparation of a cell extract Techniques for breaking bacterial cells: Physical methods :ultrasonic （超声波） Chemical methods: lysozyme（溶菌酶）/ EDTA, SDS Lysozyme: digest the polymeric in cell wall EDTA: remove magnesium ions and inhibit enzymes that degrade DNA SDS: remove lipid molecules in the cell membranes. 3.1.2 Preparation of a cell extract 3.1.3 Purification of DNA from cell extract Remove protein from cell extract: phenol (苯酚） or 1:1 mixture of phenol and chloroform（氯仿） When protein content is great in cell extract: treat with a protease（蛋白酶） ( pronase/ proteinase K) RN