Life Science Quarterly July 2000

July 2000 Genelute Kit

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 1    July 2000

 Molecular Biology Application Notes

The GenEluteTM Endotoxin-free DNA Purification Kit: Pure DNA for Endotoxin-Sensitive Applications

by Fuqiang Chen, Scott Weber and Jo Anne Kerschner
Sigma-Aldrich Corporation, St. Louis, MO, USA

Introduction

Endotoxins are lipopolysaccharides (LPS), found among outer cell-membrane components of Gram negative bacteria such as E. coli. One E. coli cell contains approximately 2,000,000 LPS molecules. In mammalian systems, endotoxins are pyrogenic, causing fever and endotoxic shock syndrome. The potential for contamination of purified plasmid preps, raised in E. coli, by endotoxins is a major concern in gene therapy. Additionally, endotoxins can significantly reduce transfection efficiencies in endotoxin-sensitive cell lines such as COS-7, HEK293, and CHO-K1.1 Other applications affected to a lesser extent are restriction endonuclease digestion, cloning, sequencing, and PCR.

LPS molecules are negatively charged like DNA and are often co-purified with plasmid DNA. Both molecules behave similarly on a silica surface or in anion exchange chromatography. The density of endotoxins in CsCl ultracentrifugation is similar to that of the plasmid-ethidium bromide complex, causing contamination of CsCl-purified DNA. Purified plasmid DNA prepared by silica-based plasmid isolation systems may contain extremely high levels (>1000 EU/µg plasmid DNA) of endotoxins.1 Plasmid DNA prepared by anion exchange chromatography or two rounds of CsCl ultracentrifugation contains lower, but still significant levels (>4 EU/µg DNA) of endotoxin.2 In order to produce material that is endotoxin-free, additional reagents and steps are required. This can be accomplished either during or after DNA extraction.

Materials and Methods

All materials were supplied by Sigma Chemical (St. Louis, MO) unless otherwise stated.

The GenEluteTM Endotoxin-free Plasmid Midiprep and Maxiprep kits provide a simple, rapid, and cost-effective method for isolating endotoxin-free plasmid DNA from recombinant bacterial cultures. An endotoxin removal step is integrated into the GenEluteTM plasmid purification system. After endotoxin removal, plasmid DNA is purified on a silica-based membrane in an easy-to-use spin-column format. Up to 120 µg of plasmid DNA free of endotoxin contamination (<0.1 EU/µg DNA determined by Limulus Amebocyte Lysate kinetic test) can be recovered from 5-40 ml of overnight bacterial culture with the Midiprep kit. Up to 1.2 mg of plasmid DNA free of endotoxin contamination (<0.1 EU/µg DNA) can be recovered from 25-130 ml of overnight bacterial culture with the Maxiprep kit.

Recombinant E. coli cells are harvested from an overnight culture by centrifugation and subjected to a modified alkaline-SDS lysis procedure to produce a cleared lysate. Chromosomal DNA and other cellular components are precipitated and removed from the lysate by centrifugation. Endotoxins are removed from the cleared lysate by extraction with a temperature-mediated two-phase separation. The blue dye in the lower organic phase allows easy location of the interface and facilitates collection of the clear upper phase containing the plasmid DNA. After the addition of a DNA-binding solution, the endotoxin-free cleared lysate is loaded onto a midi- or maxi-binding column that is seated inside a collection tube. Plasmid DNA is selectively bound onto the silica membrane when the lysate flows through the column during centrifugation. Salts and other contaminants are removed from the column by a spin-wash step. DNA is eluted in endotoxin-free water.

Results and Discussion

Plasmid DNA purified by this method is predominately in its super-coiled form. The 260/280 nm ratio is consistently between 1.8 and 2.0. There is no evidence of genomic DNA or RNA contamination (determined by agarose gel electrophoresis). The level of residual endotoxins in purified DNA is consistently <0.1 EU/µg plasmid DNA determined by Limulus Amebocyte Lysate kinetic test. Purified plasmid DNA is ready for use in cell transfection and other downstream applications, such as restriction endonuclease digestion, ligation, sequencing or PCR.

It takes approximately 95 minutes to purify endotoxin-free plasmid DNA using the Midiprep kit and 105 minutes for the Maxiprep kit. This compares favorably with other commercially available kits (Figure 1). Endotoxin-free plasmid DNA purified using the GenEluteTM Endotoxin-free kits cuts efficiently with restriction enzymes. Figure 2 shows the action of Hind III and EcoRV restriction enzymes on the plasmid DNA (pCMV-SPORT-ßgal) when 1 µg was incubated with the enzymes at 37 ºC for 2 hours. Each lane was loaded with half of a digestion.

Figure 3 demonstrates the transfection efficiency of plasmid DNA prepared with Sigma's GenEluteTM Endotoxin-free kits versus those of competitors. Plasmid DNA was prepared according to manufacturers' instructions. The data show the average of six replicates for each plasmid isolation method. All transfections were in CHO-K1 cells. The OD measurements were taken at 420 nm and the units of ß-galactosidase per plate were determined using the ß-Galactosidase Reporter Gene Activity Detection kit (ß-Gal; Product Code: GAL-A). The cells were grown to 60-75% confluency. Cells were transfected using 3 µg of plasmid DNA per 15 µl of Escort IV, a transfection reagent (Product Code: L 3287). The ß-Gal kit was used after a 68-70 hour post-transfection incubation.

Conclusions

The GenEluteTM Endotoxin-free Plasmid Midiprep and Maxiprep kits compare favorably with other commercially-available kits. Yield for the Midiprep kit is up to 120 µg and up to 1.2 mg for the Maxiprep kit. The transfection efficiency is superior compared to competitors. Purified DNA is compatible for other downstream applications such as restriction endonuclease digestion, sequencing, and PCR.

References

1. Weber, M., et al., Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. BioTechniques, 19, 930-40 (1995).
2. Cotten, M. et al., Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. Gene Therapy, 1, 239-246 (1994).
3. Birnboim, H.C., A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol., 100, 243-55 (1983).
4. Boom, R., et al., Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol., 28, 495-503 (1990).
5. Ehlert, F., et al., Importance of DNA quality for transfection efficiency. BioTechniques, 14, 546 (1993).


GenElute is a trademark of the Sigma-Aldrich Corporation.

About the Authors
Fuqiang Chen, Ph.D., is a senior scientist and Scott Weber, B.S., is an associate scientist in NAP, PCR and Sequencing R&D at Sigma-Aldrich, St. Louis, MO. Jo Anne Kerschner, Ph.D., is the manager of NAP, PCR and Sequencing R&D at Sigma-Aldrich, St. Louis, MO.
 

ORDERING INFORMATION
Product Code Product Name Unit
PLED35 GenEluteTM Endotoxin-free Plasmid Midiprep kit 1 kit (35 reactions)
PLEX15 GenEluteTM Endotoxin-free Plasmid Maxiprep kit 1 kit (15 reactions)
SUPPORTING LITERATURE
For additional information, request technical bulletins (PLED 35) and (PLEX 15) Nucleic Acid Brochure (DHB) 

 

 


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