( An Innovative Technology for the...)Introducing,,,GenElute HP,,,Endotoxin-Free Plasmid,,,Maxiprep Kit

An Innovative Technology for the Rapid Purification of Endotoxin-Free Plasmid DNA
Dave Cutter and Goodarz Goodarzi

The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit offers a novel and efficient method of isolating endotoxin-free plasmid DNA. A patent-pending endotoxin removal process has resulted in the fastest, most streamlined protocol available for purifying high-quality, endotoxin-free DNA (≤ 0.1 EU/g). This product delivers significant time-savings, overall higher yields, and better transfection efficiencies compared to anion-exchange methods.

Abstract
Endotoxins are a common contaminant in nucleic acid preparations, particularly when purifying plasmid DNA. The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit is a novel system that has been developed for the rapid purification of endotoxin-free plasmid DNA from a bacterial culture. Comparisons among the GenElute HP Endotoxin-Free Plasmid Maxiprep Kit, the GenElute HP Maxiprep Kit and other plasmid purification systems were performed. The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit sets a new standard for endotoxin-free plasmid purification while saving time and delivering higher yields.

Introduction
In typical plasmid DNA purification systems, high concentrations of endotoxins are released into the lysate. Like DNA, endotoxins are negatively charged and co-purify with plasmid DNA in both silica-based and anion-exchange-based systems. Endotoxins can also form large aggregates resulting in co-purification in size exclusion columns and cesium chloride density gradients. Therefore, both standard and traditional plasmid purification systems can result in high levels of endotoxin contaminating the purified plasmid DNA.

While not every molecular biology technique requires the use of endotoxin-free plasmid DNA, transfection of endotoxin-sensitive cell lines is a major application where endotoxin-free DNA is essential. The data shows that endotoxins reduce cell viability and transfection efficiencies among different eukaryotic cell lines, such as COS-7 and Jurkat cells. Furthermore, certain cell lines appear to be more sensitive to endotoxins, such as HuH-7 (human hepatoma) cells. Principally, endotoxins introduce a variable that can influence the outcome and reproducibility of a given transfection experiment.

The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit combines the simplicity of a bind, wash, and elute format with a proprietary solution that binds plasmid DNA to a silica matrix while preventing endotoxins from adsorbing. The technology allows for the user to consistently achieve levels of endotoxin that are less than 0.1 endotoxin units per g of plasmid DNA.

Figure 1. Flow Chart of the purification/endotoxin-removal procedure (patent pending). The cleared lysate is mixed with the Binding Solution which includes reagents that efficiently inhibit endotoxins from adsorbing to the Binding Column while promoting the adsorption of plasmid DNA. No separate step of endotoxin removal is required. Materials and Methods
Culture Preparation
Recombinant E. coli strain DH5α pCMV-SPORT-β-gal was inoculated into LB medium containing 100 g/mL of ampicillin and was grown shaking at 275 rpm for 17 hours at 37 C.

Plasmid Preparation
150 mL of bacterial culture was used for each sample and plasmid was isolated following the manufacturer's protocol.

Plasmid Analysis
Plasmid concentrations were determined by taking absorbance readings (260 nm-320 nm). Total yield (mg) was calculated by multiplying the concentration by the volume of recovered eluate. The purity of the samples was determined by calculating the ratio of absorbance (260 nm-320 nm)/(280 nm-320 nm).

Endotoxin Testing
Endotoxin levels were determined by using the QCL-1000 Quantitative Chromogenic LAL kit (BioWhittaker, Walkersville, MD, USA) following the Test Tube Method.

Results and Discussion
The GenElute HP Endotoxin-Free Maxiprep Kit was compared to three other commercially available kits by purifying pCMV-SPORT-β-gal from the same bacterial culture. The first was the Sigma GenElute HP Maxiprep Kit, the second was an endotoxin-free kit that utilizes anion-exchange gravity-flow columns, and the third was an endotoxin-reducing silicabased kit that incorporates magnetic particles into the purification process. In addition, plasmid pCMV-SPORT-β-gal was prepared from a separate culture on two cesium chloride gradients (2X CsCl banding).

Plasmid recoveries, endotoxin levels, and time/prep for all the samples were compared in Table 1. Plasmid yields obtained from the GenElute HP Endotoxin-Free Plasmid Maxiprep Kit were similar to or better than the other methods. Endotoxin levels were also among the lowest when compared to the other available endotoxin-free kits, the Sigma GenElute HP Maxiprep Kit as well as the 2X cesium chloride sample. The most significant difference between the endotoxin-free kits was in the time/prep. The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit took only 35 minutes to complete, while two other competing endotoxin-free kits took over four times as long. Only the GenElute HP Maxiprep Kit was comparable in preparation time, but the purified sample had higher levels of endotoxin.

Table 1. Comparison of plasmid yield, endotoxin levels and time/prep for the different purification systems for pCMV-SPORT-β-gal.

Purification System Plasmid Yield (mg) Endotoxin Levels (EU/g) Time/Prep (minutes) GenElute HP Endotoxin-Free Maxiprep Kit 1.4 0.02 35 GenElute HP Maxiprep Kit 1.6 7.4 30 Endo-Free Anion-Exchange-based Kit 0.7 0.04 165 Endo-Free Silica-Magnetic-based Kit 1.3 0.17 150 2X Cesium Chloride gradient 0.3 1.4 3 days

Transfection efficiencies into HuH-7 (human hepatoma) cells were compared among the different purification systems and reported in Figure 2. The results show that the sample purified using the GenElute HP Endotoxin-Free Plasmid Maxiprep Kit had the highest transfection efficiency when compared to the other commercially available kits.

Figure 2: Comparison of transfection efficiencies into HuH-7 cells using different purification systems with pCMV-SPORT-β-gal. The data show the average and standard deviations of six replicates from each sample prepared. HuH-7 cells were transfected using ESCORT II Transfection Reagent (Sigma Product Code L6037). The efficiency of the transfection was determined by measuring the b-galactosidase activity using the b-galactosidase Reporter Gene Activity Detection Kit (Sigma Product Code GALA-1KT).

Transfection of pCOP-Green-C into HuH-7 cells
Plasmid pCOP-Green-C is a mammalian expression vector that encodes the copepod green flourescence protein. HuH- 7 cells were seeded onto glass cover slips and then grown to 60-70% confluency. The cells were then transfected with 3 g of plasmid DNA using ESCORT II Transfection Reagent. Plasmid DNA was isolated from a GenElute HP Endotoxin- Free Maxiprep. At 72 hours post transfection, the glass cover slips were mounted onto microscope slides and the cells were observed under a fluorescence microscope. Panel A is a picture of the cells following transfection under bright field exposure. Panel B is the same area of cells under fluorescent exposure, which reveals a high transfection efficiency.

Summary
The GenElute HP Endotoxin-Free Plasmid Maxiprep Kit's innovative technology combined with a straightforward procedure makes this kit the fastest endotoxin-free kit available, enabling it to out-perform both current and conventional purification methods.

• High plasmid recoveries with endotoxin levels of ≤0.1 EU/g
• Only 35 minutes from pelleted cells to purified plasmid
• High-quality plasmid DNA necessary for high transfection efficiency in endotoxin-sensitive eukaryotic cell lines

For more information, visit: www.sigma-aldrich.com/molecularbiology

2005 Sigma-Aldrich Co. All rights reserved.
SIGMA, SAFC, SIGMA-ALDRICH, ISOTEC, ALDRICH, FLUKA, SUPELCO, GenElute, and ESCORT are trademarks belonging to Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology LP. GC5, and GC10 are trademarks of Gene Choice, Inc. Riedel-de Haen trademark under license from Riedel-de Haen GmbH. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip.
'"/>

Source: