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


Achieve superior transfection efficiencies with StrataPrep midiprep kit

Scott Basehore Jeff Braman
Stratagene

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, centrifuging 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 Endotoxin-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 background measurement.

Fig.1

Conclusions

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.

REFERENCES

  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.


'"/>

Source:


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:12/24/2014)... , Dec. 24, 2014   BioLife Solutions , ... manufacturer and marketer of proprietary clinical grade hypothermic ... precision thermal shipping products for cells and ... will hold its 2015 Annual Meeting of Stockholders on ... the expected date for the Annual Meeting represents a ...
(Date:12/24/2014)... Boston, MA (PRWEB) December 23, 2014 ... presentation, Dr. James L. Sherley, director of the Adult ... attention on an often overlooked and under appreciated unique ... “Asymmetric Self-Renewal by Distributed Stem Cells: Misunderstood in the ... his message to congress participants. He gave the ...
(Date:12/24/2014)... New York (PRWEB) December 23, 2014 According ... the Long Island Affiliate of ITRA Global, the national office ... but steady recovery. This is evidenced by the third ... strong stock market. Low energy costs have held inflation ... The only negative is the housing market remaining soft. , ...
(Date:12/24/2014)... 2014 The report expects global cell ... 2019. It also provides a carefully analyzed data about ... for the market. , Full Copy of Report @ ... global market for cell expansion will keep witnessing growth ... this growth to be driven by rapid technological advancements, ...
Breaking Biology Technology:BioLife Solutions Sets Date for Annual Meeting of Stockholders 2Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 2Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 3Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 4ITRA Global Reports Long Island Is Out of Sync With The National Office Market 2ITRA Global Reports Long Island Is Out of Sync With The National Office Market 3The Cell Expansion Market is Expected to Reach $14.8 billion by 2019 - New Research Report by MarketsandMarkets 2The Cell Expansion Market is Expected to Reach $14.8 billion by 2019 - New Research Report by MarketsandMarkets 3
... New Service Helps Customers to Treat More Patients, ... ARAY ), a global leader in the ... begin offering a treatment planning,service to new and ... centers to access a dedicated team of remote ...
... Quintiles,Transnational Corp. today announced the completion of ... company,s continued,growth under the leadership of founder and ... management team. Gillings and global private,investment firms Bain ... in Quintiles, with 3i also becoming a significant ...
... Ind., Jan. 23 Zimmer Holdings, Inc.,(NYSE: ZMH ... announced,today it will be participating in the Wachovia Securities ... 10:15 a.m. Eastern Time., A live webcast of ... at http://investor.zimmer.com . The webcast will,be archived for ...
Cached Biology Technology:Accuray Announces New Treatment Planning Service Offering 2Accuray Announces New Treatment Planning Service Offering 3Accuray Announces New Treatment Planning Service Offering 4Quintiles Completes Previously Announced New Investor Partnership, Co-Led by Founder, Chairman and CEO Dennis Gillings, Bain Capital, TPG and 3i 2Quintiles Completes Previously Announced New Investor Partnership, Co-Led by Founder, Chairman and CEO Dennis Gillings, Bain Capital, TPG and 3i 3
(Date:12/10/2014)... DUBLIN , Dec. 08, 2014 Research ... announced the addition of the "Biometrics Market ... http://photos.prnewswire.com/prnh/20130307/600769 ... for projects such as rural banking and upgradation ... major trends witnessed in the market. Besides the ...
(Date:12/4/2014)... GARDENS, Fla. , Dec. 3, 2014 /PRNewswire/ ... solutions, today announced that its DigitalPersona ® ... fingerprint readers have been deployed throughout First Bank ... , South Carolina and ... in Southern Pines, North Carolina ...
(Date:11/21/2014)... , Nov. 19, 2014  Earlier this year ... New York College, and one of the most prolific ... signals that are transmitted from Smartphones to third party ... Smartphones and has one of the earliest known patents ... its usage in the military, child care, elder care ...
Breaking Biology News(10 mins):Biometrics Market in Japan 2014-2018: Key Vendors are DDS, Fujitsu, Hitachi and NEC 2First Bank Partners with Crossmatch to Increase Log In Security 2First Bank Partners with Crossmatch to Increase Log In Security 3Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 2Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 3Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 4
... Athens, Ga. Companion animals that have a long-term need ... top-selling antiplatelet drug marketed to humans: clopidogrel, commonly known ... Georgia College of Veterinary Medicine have found ,that clopidogrel may ... long-term anticoagulant therapy. In addition, it may offer a ...
... Associate Professor at The City College of New York (CCNY), have ... water so that it can be scooped up like the fat ... soup. The agent could potentially be used to recover oil lost ... Professor John said. In the laboratory, Professor John and colleagues ...
... in cities, disposing of garbage and recycling anything that ... city and cell, health problems can arise when the ... of Michigan cell biologist Haoxing Xu and colleagues reveals ... recycling center, the lysosome, functions. These insights, which may ...
Cached Biology News:Plavix may be treatment for dogs at risk of thromboembolic disease 2Opening the gate to the cell's recycling center 2
... selects single cells, or clusters, with rapid, ... all, a single cell can be targeted, ... collected into the pipette, and automatically transferred ... plate. Quixells unique aspiration and expulsion system ...
... Microarrays designed for genome-wide microRNA expression ... and powerful Paraflo microfluidic on-chip synthesis ... part of our comprehensive microRNA Expression ... The Viruses microRNA Microarray contains ...
... At the forefront of Agilent analytical ... Built-in local area networking (LAN) allows you ... sites--for fast and informed decision making. Just ... has all the flexibility and performance needed ...
The novel and unique HCTplus delivers unmatched information-rich data-dependent LC/MSn data for todays challenge in proteomics and metabonomics work....
Biology Products: