Plasmid Purification

This paper describes a new method o5 plasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or e th id ium bromide, costly ultracentri /ugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel fi l tration chromatography, at low pressure (1 bar) or medium pressure (between 5 and I0 ba r s ) , using Sephacryl S1000 or 5uperose 6B. It pe rmi t s r e cove ry oI plasmids : (I) in preparative quantities (from 300 gg to # rag), (II) exempt from RNA, DNA and protein contamination, and (Ill) suitable for various common gene t i c engineering procedures immediately after purification. To test the reliability of the technique as well as the degree o5 purilication, the plasmids were used to construct thermoampliIiable vectors, carrying the tacUV5 promoter and the 5' end of the 8-galactosidase gone with a single EcoRl site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.