Navigation Links
Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell,,,Line

Achieve superior transfection efficiencies with StrataPrep midiprep kit

Scott Basehore Jeff Braman

Plasmid DNA was purified with Stratagenes StrataPrep EF plasmid midiprep kit, a kit based on anion exchange chromatography, magnetic beads, and cesium chloride density gradient centrifugation. Plasmid yields and purity (as judged by 260-nm to 280-nm optical density ratios) were comparable with the four methods. However, endotoxin levels of the StrataPrep kit- and ion-exchange-purified plasmid DNA were nearly 800 times lower than magnetic bead-purified DNA and three times lower than DNA purified by cesium chloride density gradient centrifugation. The StrataPrep-purified plasmid yielded the highest efficiency transfection of an endotoxin-sensitive cell line when compared to the other three methods.

The StrataPrep EF plasmid midiprep kit is a simple and convenient method for obtaining up to 350 g of high-purity plasmid DNA from a single midi-spin cup device.1 Contaminating endotoxin may be easily removed by using the kits unique endotoxin removal buffer (ERB). Endotoxin, a common contaminant of plasmid DNA isolated from gram-negative E. coli hosts, is difficult to remove2; its presence in plasmid preparations lowers the transfection efficiency of endotoxin-sensitive cell lines2,3 and complicates the interpretation of experiments designed to examine signal transduction pathways triggered by a measured addition of endotoxin.4 Stratagenes unique ERB contains a color indicator to monitor endotoxin removal, thereby facilitating the purification process. Following endotoxin removal, plasmid DNA is bound to a special glass fiber matrix in a StrataPrep Kit midi-spin cup. Contaminants are removed by adding wash solution to the spin cup, centrifugi ng the cup, and eluting the purified DNA from the matrix in low-ionic-strength buffer. The resulting purified plasmid DNA is ready for subsequent molecular biology applications.

Four different plasmid purification methods were analyzed. Stratagene's StrataPrep EF plasmid midiprep kit, Qiagen's QIAfilter Plasmid Maxi kit with additional Endotoxin Free Buffer set (AEC: anion exchange chromotography), Promega's Wizard PureFection Plasmid DNA Purification system (MB: magnetic beads), and cesium chloride density gradient centrifugation (CsCl).

Yields and Purity

pCMV b-gal plasmid DNA was isolated from Epicurian Coli XL1-Blue MRF E. coli cells with the StrataPrep kit. Yields of plasmid DNA ranged from 360 g using the MB method to 473 g for CsCl. Yields of the StrataPrep Kit and AEC-purified DNA were 392 g and 433 g, respectively, for an equivalent volume of bacterial culture (Table 1).

The 260-nm to 280-nm optical density (OD) ratios ranged between 1.8 and 1.9 for each plasmid preparation (Table 1), indicating a high level of purity.5 OD values at 240 nm were also measured to detect other contaminants such as EDTA6 and detergents. The 260-nm to 240-nm OD ratios ranged between 1.5 and 1.7, which indicated that the plasmid preparations were essentially devoid of these contaminants6 (Table 1).

Endotoxin units per microgram of plasmid DNA (EU/g) were measured (BioWhittaker, Inc.) using the Kinetic QCL assay. Endotoxin levels for the StrataPrep Kit and AEC-purified DNA were approximately 800 times lower than MB-purified DNA and 3 times lower than CsCl-purified plasmid DNA (Table 1).

Transfection of Endo toxin-Sensitive Cell Line

The transfection efficiency for each plasmid preparation was determined by exposing 7.5 x 106 endotoxin-sensitive RAW 264.7 cells4 to calcium phosphate precipitates containing 15 g of StrataPrep kit, AEC-, MB-, and CsCl-purified pA3-3.4Luc (IL-1Ra promoter/luciferase reporter) plasmid. Luciferase expression in this plasmid is stimulated by endotoxin. Cells were exposed to plasmid-calcium-phosphate precipitates for 5 hours, shocked in glycerol for 90 seconds, and washed. Cells from each plasmid transfection were divided equally into a set of six plates and returned to the incubator.

Forty hours post-transfection, three plates of each plasmid preparation set were treated with an excess (1 g/ml) of E. coli lipopolysaccharide (LPS), while the remaining three plates of each set were not stimulated. After an additional 8 hours of incubation, LPS-stimulated and unstimulated cells for each plasmid preparation were harvested, lysed, and assayed for luciferase activity and total protein. Relative light units (RLU) were normalized to protein concentration and expressed as RLU/mg of protein.

The transfection efficiency of StrataPrep-purified DNA, measured as total RLU/mg of protein for stimulated cells, was 1.3-, 3.4-, and 2340-fold higher than stimulated cells transfected with CsCl, AEC, and MB DNA, respectively (Figure 1). The fold RLU increase over background (RLU LPS-stimulated divided by RLU LPS-unstimulated control) for StrataPrep Kit-, CsCl-, AEC-, and MB-purified plasmid was 29, 18, 12, and 0.16, respectively. The high induction ratio for StrataPrep DNA reflects both a high transfection efficiency (indicated by enhanced LPS stimulation compared with the other preps) and negligible degree of endotoxin contamination, evident from the low LPS-unstimulated bac kground measurement.



Purification of plasmid DNA with the StrataPrep kit is simple to perform and generates high yields of pure DNA. Because endotoxin is effectively removed, high-efficiency transfection of endotoxin-sensitive mammalian cells is easily achieved.


  1. Basehore, S. and Braman, J. (1999) Strategies 12: 5-7.

  2. Cotton, M., et al. (1994) Gene Therapy 1: 239-246.

  3. Weber, M., et al. (1995) BioTechniques 19: 930-939.

  4. Sweet, M. and Hume, D. (1996) J. Leukocyte Biol. 60: 8-26.

  5. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

  6. Held, P. (1998) Pharmaceutical Lab. February: 4-6.



Page: All 1 2 3 4

Related biology technology :

1. Highest Possible Transformation Efficiencies for High-Throughput Applications
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
4. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
5. Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal Fibroblasts
6. Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
7. Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells
8. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
9. High-Efficiency Transfections Achieved with New Low-Toxicity Reagent
10. Efficient Transfection of Neurospora Crassa
11. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
Post Your Comments:

(Date:11/27/2015)... (PRWEB) , ... November 27, 2015 , ... ... Program that includes over 2,000 technical presentations offered in symposia, oral sessions, ... and applied spectroscopy, covers a wide range of applications such as, but not ...
(Date:11/26/2015)... November 26, 2015 ... Accutest Research Laboratories, a leading ... Organization (CRO), has formed a strategic ... - Temple Health for joint work ... (Photo: ) , --> ...
(Date:11/25/2015)... , November 26, 2015 ... Global Biobanking Market 2016 - 2020 report analyzes ... maintaining integrity and quality in long-term samples, minimizing ... long-term cost-effectiveness. Automation minimizes manual errors such as ... technical efficiency. Further, it plays a vital role ...
(Date:11/25/2015)... 2 nouvelles études permettent d , identifier ... souches bactériennes retrouvées dans la plaque dentaire des ... Ces recherches  ouvrent une nouvelle voie ... l,un des problèmes de santé les plus fréquemm ... --> 2 nouvelles études permettent d , ...
Breaking Biology Technology:
(Date:11/18/2015)... Nov. 18, 2015  As new scientific discoveries deepen ... and other healthcare providers face challenges in better using ... patients. In addition, as more children continue to survive ... adulthood and old age. John M. Maris, ... Hospital of Philadelphia (CHOP) . --> ...
(Date:11/17/2015)... PARIS , November 17, 2015 ... November 2015.   --> Paris from ... --> DERMALOG, the biometrics innovation leader, has invented the ... and fingerprints on the same scanning surface. Until now two ... fingerprints. Now one scanner can capture both on the same ...
(Date:11/17/2015)... 17, 2015  Vigilant Solutions announces today that Mr. ... of Directors. --> --> ... from the partnership at TPG Capital, one of the ... $140 Billion in revenue.  He founded and led TPG,s ... TPG companies, from 1997 to 2013.  In his first ...
Breaking Biology News(10 mins):