A midi-prep double-stranded DNA isolation has been developed to generate a sufficient amount of template DNA for several Sequenase[TM] catalyzed fluorescent terminator reactions. Here, one bacterial colony which harbored the plasmid of interest is picked into 3 ml of liquid media containing ampicillin and incubated in a 37degC shaker for 8-10 hours. At this time, the culture is transferred into 50 ml of ampicillin-containing media and incubated further for 10-12 hours. After harvesting the cells by centrifugation, a diatomaceous earth-based alkaline-lysis purification method (19-22) is performed, similar to that discussed above for large scale DNA isolation. The purified DNA is crudely assayed for concentration and purity by agarose gel electrophoresis against known standards. The approximate yield of double-stranded DNA using this method is 1 ug of DNA per ml of cell culture. For a 50 ml cell culture, about 50 ug of DNA are recovered, and 5 ug are used typically in a Sequenase[TM] terminator reaction.
Note: This procedure is the method of choice for isolating double stranded plasmid-based templates for the Sequenase Dye-Labeled Terminator Sequencing Reactions.