Foundation and Development of a Large-scale Production Procedure for High-purity Plasmid DNA
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Abstract:
The influential factors in the plasmid DNA extraction on the quality of the plasmid DNA was investigated, the extraction conditions of plasmid DNA were optimized and the quality-controlling standards were set in order to produce the high-purity plasmid DNA. Effect of temperature inducement on the production of plasmid DNA was studied by increasing the temperature from 37 ℃ to 43 ℃ at intervals of 1 ℃. After 12 hours of cultivation at 42 ℃, the plasmid yield was significantly increased by 66%, which demonstrated that the new method could be used for economically preparation of recombinant plasmids. Moreover, the standard alkaline lysis method for preparation of plasmid DNAs was modified and the optimized procedure was as follows: removing the cell fragments and the epactal proteins by LiCl; precipitating the plasmid DNA by PEG8000 to wipe off the digested RNA; and removing the reliquus protein by Phe-nol-CH3Cl3.In order to obtain the high-purity plasmid DNA, the crude plasmid DNA was further purified by different kinds of chromatography.
