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Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A

termine whether bacterial density affected the quality or quantity of plasmid DNA isolated, we tested the effect of using different ODml quantities of bacteria in Aurum minipreps. We found that plasmid yield increased until the maximum recommended load of bacterial culture (12 ODml) was reached (Table 4). In all cases, the plasmid yield was lower than was normally attained with cells grown in LB Amp medium, and we attribute this to using 2x YT medium. Using 50% or 100% more culture than recommended reduced the plasmid yield without greatly affecting the quality of the preps (Figure 2). We concluded that for Aurum minipreps, optimum quality and quantity of plasmid are obtained using 12 ODml of cells grown in LB Amp. Henceforth, LB Amp was used exclusively as a growth medium.

We were interested in testing how the new Aurum kits compared with other plasmid purification kits commonly used in our laboratory. We found that plasmid yields using the other kits were dramatically lower than what we achieved from similar cultures with the Aurum plasmid kits (Table 5). Aurum preps were of high quality, equivalent or better than those obtained from other kits (Figure 3, Tables 5 and 6).

We tested the quality of isolated plasmid DNA by four criteria: restriction digests, DNA sequencing, and bacterial or yeast transformations. All kits tested gave plasmid DNA that was digested equivalently by restriction enzymes (Figure 4). Two plasmid samples gave poor or unusual restriction digestion patterns (Figure 4, lanes 3 and 11), and both of these plasmids were isolated from endA+ bacterial strains. We noticed no differences in the ability of isolated plasmids to transform either bacteria or yeast.
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