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
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
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