质粒 dna（质粒 dna）.doc

质粒 dna（质粒 dna） Bacterial plasmids are double stranded, closed loop DNA, small range from 1KB to 200KB above the plasmids are genetic components ranging from autonomous replication in the cytoplasm, independent of cell chromosome outside, usually sustainable and stable in a free state staining in vitro, but under certain conditions can also be integrated into the reversible the host chromosome, and replication with replication of chromosomes, and transmitted to the offspring through cell division. Plasmids have become the most commonly used vectors for gene cloning, and the important condition is that a large number of purified plasmid DNA molecules can be obtained. At present, many methods have been used for the extraction of plasmid DNA. The plasmid DNA was extracted by alkaline lysis. The alkaline lysis method is the method for preparation of plasmid DNA for a wide application, the basic principle is: when the cell in NaOH and SDS solution in the cleavage, protein and DNA denaturation, when adding neutralizing liquid after plasmid DNA molecules can rapid renaturation in dissolved state, centrifugation left in the supernatant protein; and chromosome DNA invariant, cottony, centrifugal can settle down. The method of purifying plasmid DNA usually uses plasmid DNA relatively small and covalently closed loop two properties. For example, CSCL ethidium bromide gradient centrifugation, ion exchange chromatography, gel filtration chromatography, polyethylene glycol precipitation method, but these methods are relatively expensive or time-consuming. The DNA plasmid minipreparation, after phenol, chloroform, RNA enzyme digestion and ethanol precipitation of simple steps to remove residual protein and RNA, plasmid DNA has been purified and can satisfy the requirements of bacterial transformation, separation and enzyme digestion, conventional DNA fragment subcloning and labeling, so in the laboratory of molecular biology in common. Preparation of reagents 1. solutions I: 50mM g