Cultures should be inoculated from a single colony and grown in the presence of an appropriate antibiotic for the duration of the culture. Sub-culturing can be performed from starter cultures, but results may vary. Cells should be harvested as the culture transitions from logarithmic to stationary phase to ensure the greatest quantity of DNA is present with little cell lysis occurring. In routine cloning workflows with medium to high-copy plasmids, a single colony can be used to inoculate a 5-10 ml culture grown at 37°C for 12-16 hrs. Processing 1-3 ml of this culture should yield between 3-6 μg of a high copy plasmid or 1-2 μg of a medium copy plasmid. Lower copy plasmids will produce lower yields, which can be addressed by processing larger culture volumes with concomitant scaling of the B1-B3 buffers or by concentration of the purified DNA.
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