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Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A

Audra Day, Colette Schneider, Lisa Ottmers, and Brandt L. Schneider Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.


Introduction
Small-scale isolation and purification of plasmid DNA from E. coli bacterial strains is a widely used and essential technique in molecular biology laboratories. With the rapid pace of molecular research, it is vitally important that plasmid DNA isolation and purification protocols and commercially available kits satisfy four criteria they should be reliable, efficient, reproducible, and cost-effective. We conducted an extensive test of Bio-Rads new Aurum miniprep and midiprep plasmid DNA isolation kits. We conclude that these kits satisfy all of the above criteria.


Methods
Bacterial strains were transformed with 1 l of 10 pg/l of various plasmids by electroporation (Bio-Rad Gene Pulser electroporator), and plated to LB plates containing 100 g/ml ampicillin (Amp). Single colonies were grown to saturation overnight in 3 ml of liquid LB containing 100 g/ml Amp for minipreps, or 30 ml of the same medium for midipreps. Densities of the cultures were determined according to the Aurum protocol, and 12 ODml* or 50 ODml of each culture were used for plasmid purification for miniprep and midipreps, respectively. Plasmid concentrations were quantitated with Bio-Rads VersaFluor fluorometer and Hoechst dye 33258 DNA quantitation kit. Plasmid quality was assessed by four criteria: restriction digests, transformation efficiency with both bacterial and yeast hosts, and sequencing. Restriction digests were done on 500 ng of plasmid in 20 l with EcoRI (NEB) for 2 hr at 37C. Digests were resolved on 0.7% agarose TAE gels. Bacteria were transformed as described above. Yeast were transformed by either a standard lithium acetate protocol (Gietz and Woods 1994) or by electroporation with 100 ng of plasmid. DNA sequencing was performed directly on 100 ng of plasmid with 1 pmol of primer by a centralized core facility under standard conditions (available on request).

*Bacterial Growth Guidelines
Aurum plasmid mini and midi kits can process up to 12 ODml and 50 ODml, respectively, of bacterial culture grown in a variety of different broths, such as LB (Luria-Bertani broth), LBG (LB + 2% glycerol), TB (Terrific Broth), and 2x YT. The quantity of bacteria processed in a purification procedure, measured in ODml, was determined as follows:

First, a sample of culture was diluted 1:20 by combining 50 l of bacterial culture with 950 l of sterile broth. The optical density of this sample at λ = 600 nm (OD600) was multiplied by 20 to calculate the density of the original undiluted culture.

Depending upon the OD600, a specific volume of the undiluted culture was selected to provide an appropriate quantity of bacterial cells for processing. Appropriate volumes of culture were calculated using the following equation:

(OD600 of undiluted culture) x (culture volume in ml) = #ODml

For example, 12 ODml of bacteria would require 2 ml of an undiluted culture with an OD600 of 6, or 4 ml of a culture with an OD600 of 3.

1 OD600 is equivalent to approximately 8 x 108 cells/ml


Results
A wide variety of E. coli strains are frequently used for the propagation of plasmids. We have found that certain bacterial strains and certain plasmid purification kits can result in poor plasmid yield or poor plasmid quality. Therefore, we assessed the yield and quality of plasmids isolated from 14 common laboratory E. coli strains with the new Aurum miniprep and midiprep kits. We tested both endA- and endA+ bacterial strains because both the quality and quantity of plasmid DNA isolated from endA+ strains can be poor. The yields from these experiments are summarized in Tables 1 and 2. On average, ~20 g and 120 g of plasmid were isolated from minipreps and midipreps, respectively. The yields from minipreps varied more than from midipreps, but this may be because more variables (strains, endA- vs. endA+) were tested. In general, yield correlated most closely to specific plasmid type. Notably, good yields of plasmid could be obtained from endA+ strains.

Bacterial growth media can affect the quality and quantity of isolated plasmid DNA. At least four different media are commonly used for the growth of E. coli. We routinely use LB Amp but were interested in comparing the plasmid yield and quality when LBG Amp, 2x YT, or TB Amp was used. We found that plasmid yield seems to vary considerably with the type of medium used (Table 3). Notably, LBG Amp medium gave poor yields and plasmids that migrated unusually on agarose gels (Table 3, Figure 1). LB Amp and TB Amp gave the best yields and highest quality of plasmid.

To determine 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. Plasmids from all kits and conditions transformed strains equivalently with high efficiency. However, we did notice differences when we sequenced plasmids derived by different purification methods (Table 6). Notably, Aurum preps performed the best under these conditions.


Conclusions
Purified plasmid DNA from the new Aurum miniprep and midiprep kits was of high quality and reproducibly performed very well in restriction digestions, bacterial and yeast transformations, and automated fluorescent DNA sequencing reactions. The Aurum kits also equaled or outperformed other commercially available plasmid purification kits for overall yield and quality of plasmid.


Reference
Gietz RD and Woods RA, High efficiency transformation with lithium acetate, In Johnston JR (ed), Molecular Genetics of Yeast: A Practical Approach, Oxford University Press, New York, 121134 (1994)

DH5α and DH10B are trademarks of Life Technologies. QIAprep is a trademark of QIAGEN. Strataprep, XL1-Blue, XL2-Blue, and XL10-Gold are trademarks of Stratagene. Wizard Plus and SV Plus are trademarks of Promega.


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