Plasmids microscale isolation

Microscale Isolation of Plasmid DNA by Alkaline Lysis (Bimboim, 1983)... 

As an alternative to the boiling method, students may perform the microscale isolation method. This version is more convenient to set up and complete, especially if facilities are limited. The procedure works well with unamplified or chloroamphenicol-amplified cultures. Each milliliter of an unamplified, overnight culture will yield approximately 1-2 /xg of plasmid DNA. This isolation procedure requires IV2 to 2 hours of student lab time if a cell culture is prepared in advance. 

Extrachromosomal DNA molecules called plasmids are harbored in some strains of E. coli. The normal copy number of the plasmids is small, between 2 and 10 however, if these strains of E. coli are grown in the presence of chloramphenicol, up to 3000 copies may be replicated per cell. Plasmid DNA has been demonstrated to be a useful vehicle in molecular cloning. This experiment describes a method for the growth of E. coli and amplification of the ColEl plasmids. The plasmids will be isolated from E. coli cells by one of two methods, a large-scale boiling method or a microscale alkaline lysis method. The DNA plasmids will be measured for molecular size by agarose electrophoresis. 

Two isolation procedures based on methods in category 2 are described in this experiment, a large-scale and a microscale method. Each procedure yields plasmid DNA that is sufficiently pure for size analysis by agarose electrophoresis and for digestion by restriction enzymes as described in Experiment 15. 

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