转化（transformation）转染（transfection)大肠杆菌捕获噬菌体DNA.PPT

二乙氨乙基（diethyl-aminoehtyl，DEAE）葡萄糖能促进哺乳动物细胞摄入外源DNA。 The negatively charged DNA binds to DEAE and the complex is taken up by the cell via endocytosis. 4. DEAE-dextran (DEAE---葡萄糖传染法) 吸附到细胞表面后被细胞吞入，部分DNA可以进入到细胞核里。 DEAE-dextran 细胞 外源DNA 预处理 摄入 DEAE-dextran 外源DNA 细胞 混合 DEAE-dextran is toxic to cells! 5. Via virus 18 Recombinant DNA Technology Dr. Hui LI Office : S408 Tel:Topic 3 Transfer of target gene into receptor cell Bacteria that are capable of uptaking the foreign DNA are called competent; To increase the permeability of cell membrane. How to make competent E.coli cells? Procaryote cells as receptor cells Use restriction enzymes-deficiency E.coli Species Calcium chloride (CaCl2)transformation is a method of promoting competence for bacterial cells. Principle Procedure E.coli culture Until OD600 0.3-0.4 On ice 5-10 min Collection of bacterial cells by centrifugation at 4℃ Suspension of cell pellet with chill CaCl2 Collect cells by centrifugation at 4℃ Store at -70 ℃ On ice 30 min Suspension of cell pellet with chill CaCl2 Collect cells by centrifugation at 4℃ Suspension of cell pellet with chill CaCl2 How to introduce foreign DNA? （1）转化（transformation） （2）转染（transfection) 大肠杆菌捕获噬菌体DNA的过程。 Methods of introduction foreign DNA into competent cells （3）转导（transduction) 借助噬菌体把外源DNA导入细菌的过程。 大肠杆菌捕获质粒DNA的过程。 Number of colonies / ?g DNA An excellent preparation of competent cells will give ~108 colonies per microgram of plasmid. Methods of transformation Efficiency 简单，但转化效率不高（106-108/?g DNA）。 （1） Heat shock(热休克法） 转化效率高（高于109/?g DNA）。 （2）Electroporation(电转化法) 10ng vector 100?L competent cells On ice, 10 min 42oC 30s-120s Add 1mL LB culture Incubate at 37oC for 1h 10-100 ?L plated on antibiotic selective media (Only transformants can survive) Adhesion of DNA Uptake of DNA （1）Heat shock Electroporation is another method of promoting competence. In the method the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell