重组DNA的转与筛选.docVIP

第四章 重组DNA的转化与筛选 　　载体与目的基因连接后，体系中可能存在下列分子：　　　a. 未连接的载体分子　　　b. 未连接的DNA片段　　　c. 载体的自连　　　d. 含有错误插入片段的重组DNA分子　　　e. 重组DNA分子　　转化过程中，这些分子同时被转移到宿主细胞内。未连接的分子即使被细菌摄取，只有在特殊的条件下才能复制，寄主的酶可降解这些DNA片段。自连的载体和错误插入的重组质粒可以进入宿主细胞进行正常的复制。因此，需要将重组克隆鉴定出来。 第一节 细菌的转化方法 　　自然界中，转化并不是细菌或的遗传信息的主要方式，实验室中只有小部分的细菌可以很容易的转化，进一步的研究发现，这类细菌含有DNA结合和吸收的代谢机制。正常条件下，大部分细菌包括大肠杆菌，只能吸收有限的DNA，为了提高转化效率，建立了一系列的转化方法：　　　a. 热激法转化感受态细胞 　　　b. PEG介导的原生质体转化 　　　c. 高压电穿孔法转化 　　　d. 显微注射法转化 　　　e. 接合转化　　　f. 噬菌体转染 表4—1 大肠杆菌常用转化方法的比较 转 化 方 法 化 效 Ca 诱 导 107-8 105-6 噬菌体转化 107-8 电 穿 孔 法 106-9100Kb） 合 转 化 104-5 center pin checks and anchoring the inner cylinder. According to While coupling alignment should keep oil out of cavity and locate the rotor axis neck raised are within the prescribed limits, there should be no gaps, Horn, Horn and Horn 0.05mm feeler gauges fit into. Center coupling, condenser water filling and water-filled according to factory requirement. For coupling three supported rotor Center, the mouth under the coupling between the value should be as stipulated in the manufacturer, usually 0.15~0.25mm. In the case of coupling connection, if possible, lifting only one bearing end of the support bearing of the rotor, remove the bearing, fitted with special tiles and special lifting tools, disc rotors, check the journal flutter tile, should generally be less than 0.10MM. When the couplings and rotor comes when there is a conflict, the coupling Centre shall prevail. 6.4.5.2.2 shaft looking for: ... 6.4.5.3. Provisional bolt confirmed the positioning of the rotor size, as well as the coupling between the adjustment shim thickness accuracy, drawings to meet the technical requirements. Plane, cylindrical and clean the coupling bolt holes should be clean and without burrs, scrap iron and grease-free, relative position of the coupling according to the drawing requirements are. Coupling top screws, round distance apart, indicators to monitor movement of the aircraft, its value is allowed to pass t