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Comparison of Growth Techniques and Media for the Purpose of Plasmid ,,, Isolation from E. coli Using the Eppendorf ,,, Perfectprep Plasmid Mini ,,, Kit

-lid tubes (Eppendorf LidBac 2.0 ml tube; 1.5 ml culture) and in 15 ml conical tubes (1.5 ml culture) with the aforementioned media.36 Experiments were carried out in parallel to ensure a direct comparison. To guarantee the initial bacteria concentration was identical in all experiments, a starter culture was grown in LB media overnight. After this initial growth, 10 l of the culture was inoculated at mid log phase (A595 = 0.643) in 1.5 ml of each media.

The Eppendorf Thermomixer R (equipped with 2.0 ml Thermoblock) was used for incubation of the LidBac tubes on the bench top after inoculation with respective media. The incubation period was 8 hours on the Thermomixer R at 37C with a mixing speed of 1,400 rpm. The 15 ml culture tubes were also inoculated with 10 l of culture and grown in a standard orbital shaker for 8 hours at 37C with a shaking speed of 400 rpm.

Spectrophotometric readings (A595) were performed using Eppendorfs BioPhotometer7 every 2 hours for a period of 8 hours (T = 0, 2, 4, 6, 8). All experiments were performed in triplicate.

E. coli Plasmid DNA isolation

The Perfectprep Plasmid Mini Kit was used to isolate and purify the plasmid DNA from E. coli. Cells were lysed using the optimized Alkaline Lysis protocol detailed in the Perfectprep Plasmid Mini Kits. After neutralization of the cell lysate, binding matrix was added to the lysate for recovery of plasmid DNA. The plasmid DNA, bound to the matrix, was then submitted to a single wash for the removal of contaminants and eluted with 70 l of Molecular Biology Grade Water8 as described in the protocol.

Results and Discussion

Comparison
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