Isolation and purification of plasmid DNA from bacteria involves three steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of plasmid DNA. Growth of the bacterial cultures is a key factor in obtaining high quality DNA. Using Eppendorfs Thermomixer R in conjunction with Eppendorfs LidBac is an ideal combination.1,2 The LidBac provides excellent gas interchange through a 0.22 m hydrophobic membrane while preventing contamination. The LidBac also allows for growth in standard microcentrifuge tubes, enhanced by excellent heat transmission of the Thermomixer R and suitable Thermoblock.
This paper will present an alternative method of growing bacteria in a more reliable manner and at a lower overall cost due to the direct use of the culture vessel in the isolation procedure. The LidBac and Thermomixer R is a dependable combination that provides researchers with an easy-to-use system to grow bacterial cultures directly on the bench top in small quantities.
Materials and Methods
Cell Cultivation and Culture Medias
Escherichia coli (E. coli) strain DH5a containing Plasmid pCS2++ were inoculated into 1.5 ml of LB (Luria-Bertani Medium), TB (Terrific Broth) and 2X YT and cultivated in membrane-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 of Bacterial Cultures in 15 ml Conical Tubes and LidBac
Figure 1. Comparison of E. coli cultivation in LB, 2X YT and TB mediums using 15 ml tubes and LidBac. A. Growth curves of bacterial cultures in LB media. B. Growth curves of bacterial cultures in 2X YT media. C. Growth curves of bacterial cultures in TB media.
In the experiments shown, the culture was allowed to proceed to the log phase. Figure 1 shows a comparison of cultures grown in 15 ml culture tubes and the Eppendorf LidBac in three different media. In all cases, growth curves of bacterial cultures were higher in the samples grown in LidBac. Clearly, the cultures experienced better growth when LidBac was used as the culture vessel, regardless of the media.
The table in Figure 2 demonstrates that the growth rates achieved were directly proportional to the yield of plasmid DNA after purification. A 1.4- to 3-fold increase is seen in these samples, depending on the media used. As expected, the greatest differences were seen in the samples grown in TB, which also had the greatest differences in growth curves (Figure 1C).
One of the critical factors related to the difference in growth rates is the superior air exchange characteristic of the hydrophobic 0.22 m membrane of LidBac. In contrast, the 15 ml culture tubes require loose capping to prevent contamination, thus limiting air exchange within the tube. Increased aeration and improved growth rates seen with the LidBac culture can also be attributed to the increased agitation rate that can be achieved with the Thermomixer R compared to conventional incubators and shakers.
Plasmid DNA Quantity Purified from Bacterial Cultures
Figure 2. Comparison of plasmid yields in the different media and culture vessels.
After data collection and analysis of a series of experiments comparing bacterial growth in different media using 15 ml culture tubes versus the Eppendorf LidBac, it can be concluded that the Eppendorf Lidion with the Thermomixer R and a rich media is a more efficient and effective method for growing small volumes of bacteria.