Preparation of Plasmid DNA
A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods basically involve three major steps:
Growth of the bacterial cell Harvesting and lysis of the bacteria Purification of plasmid DNA
The plasmid preparation, we have the plasmid DNA from large amount of bacterial chromosomal DNA. Several methods are available for removal of the bacterial DNA during plasmid preparation.
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General method
During cell lysis, the shearing of chromosomal DNA is very less; hence the large size of the chromosomal DNA tends it to be removed along with the cell debris by centrifugation. There is another method on the basis of conformational difference between plasmids and bacterial DNA. Before going to this you must prepare a clear lysate (plasmid, DNA) by centrifugation in which plasmid and DNA are present. Most plasmid resides in supercoil state.
For the separation, two different methods are commonly used: 1. alkaline Denaturation method 2. EtBr-CsCl (cesium chloride) density gradient centrifugation
When a bit of acid (acidic potassium acetate) is added, it neutralizes the base.
The basic concept of this method is to maintain a very narrow pH range in which the non supercoiled DNA will be denatured but not the supercoiled plasmid. Initially the pH of the solution containing both is adjusted at 1212.5. This in turn denatures the open circular DNA or the non supercoiled DNA
At neutral pH, the genomic DNA renatures and is trapped in the SDS/lipid/protein precipitate and forms a lump. The plasmid DNA renatures into double stranded molecules that remain in the solution
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EtBr-CsCl (cesium chloride) density gradient centrifugation
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同时具有遗传讯息及构形的 RNA 分子
Central Dogma 的演化进程
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Amino acid sequences (protein)
最后形成目前的细胞模式
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