基因工程CHAPTER 2-1PPT.ppt

* * * * * * * * * * * * * * * * * * Northern blotting Northern blotting is use a labeled probe to detect RNA complementary Colony/plaque screening Dot blot Denatured DNA is simply dotted on membrane and hybrid with probe. Western blotting The term ‘western’ blotting (Burnette 1981) refers to a procedure which does not directly involve nucleic acids, but which is of importance in gene manipulation. It involves the transfer of electrophoresed protein bands from a polyacrylamide gel on to a membrane of nitrocellulose or nylon, to which they bind strongly. The bound proteins are then available for analysis by a variety of specific protein–ligand interactions. Most commonly, antibodies are used to detect specific antigens. Lectins have been used to identify glycoproteins. In these cases the probe may itself be labelled with radioactivity, or some other ‘tag’ may be employed. There are several different method to label a probe for western. Iodinized Ab with I125; Enzyme-linked Ab or florescent labeled probes. Technique threeBacteria Transformation Recombinant molecule should be put into a host to propaganda or expression. E.coli is the first bacteria to host the foreign DNA. Mandel and Higa (1970) found that treatment with CaC12 allowed E. coli cells to take up DNA from bacteriophage λ. A few years later Cohen et al. (1972) showed that CaC12-treated E. coli cells are also effective recipients for plasmid DNA. Transformation (转化)definition: Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. Competent cell(感受态细胞): Bacteria which are able to uptake DNA are called “competent”. The competent cell can inducid by treatment with calcium chloride in the early log phase of growth. How to transform E.coli 1. conventional Competent cell transformation 2. Electroporation Electroporation power supplier and cuvettes 3.Transfomation via artificial virus transfection DNA is recombinated into a bacterial virus like lambda and be packaged