碱裂解法抽提质粒dna（Extraction of plasmid DNA by alkaline lysis）.docVIP

碱裂解法抽提质粒dna（Extraction of plasmid DNA by alkaline lysis） Experiment 1. Extraction of plasmid DNA by alkaline lysis [experimental principle] Plasmids are small, double stranded, ring-shaped DNA found in vitro, ranging in size from 1-200kb to autonomously replicating in host bacteria. In the host cell plasmid copy number is different, one is the low copy number, each cell contains only one or several plasmid molecules, known as the stringent plasmid replication, another kind of high copy plasmid copy number can reach more than 20, this type is called plasmid relaxation type copy. Plasmids can encode a number of genetic traits, such as resistance to drugs (ampicillin, tetracycline, etc.), which can be used to screen host bacteria or recombinant bacteria. Plasmid as a vector of gene engineering must have the following conditions (1) replicon (ORI): a DNA sequence with special structure; (2) one or more to facilitate the detection of genetic phenotype, such as drug resistance, color phenotype reaction; (3) one or several restriction endonuclease site for foreign gene insertion; (4) the appropriate copy number. Preparation of plasmid vectors is a routine technique in molecular biology. 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 utilizes two properties of plasmid DNA, relatively small and covalently closed loop. 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 miniprep