( ...)EndoFree Plasmid Kits

For purification of endotoxin-free ultrapure plasmid or cosmid DNA

Features and benefits

• Less than 0.1 EU/g plasmid DNA
  
• Yields of up to 10 mg plasmid DNA
  
• Greater than 90% recovery of plasmid DNA
  
• Fast and simple procedure

* Actual yields depend on plasmid copy number, size of insert, host strain, culture medium, and culture volume.
Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium.
For purification of low-copy plasmids and cosmids, EndoFree Plasmid Mega Kits are a better choice than EndoFree Plasmid Giga Kits, due to the large culture volumes required and the limited capacity of the QIAfilter Mega-Giga Cartridge

Background

Endotoxins, also known as lipopolysaccharides or LPS, are cell-membrane components of Gram-negative bacteria such as E. coli (see figure "Schematic diagram of the envelope of E. coli."). Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines (see figures "Plasmid Purification Method vs. Transfection Efficiency"and "Plasmid Purity vs. Transfection Efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for free transfection reagent. Endotoxins also induce nonspecific activation of immune responses in immune cells such as macrophages and B cells, which can lead to misinterpretation of transfection results. These responses include induced synthesis of proteins and lipids such as IL-1 and prostaglandin. Overall, endotoxins represent a non-controllable variable in transfection experiment setup, influencing the outcome and reproducibility of results and making them difficult to compare and interpret. In gene therapy research, endotoxins can interfere by causing endotoxic-shock syndrome and activation of the complement cascade.
EndoFree principle

The level of endotoxin contamination in purified plasmid DNA depends on the purification method used (see table "Endotoxin levels in plasmid preparations"). Silica-slurrypurified DNA exhibits extremely high endotoxin levels. QIAGEN, QIAfilter, and HiSpeed Plasmid Kits and 2x CsCl ultracentrifugation yield very pure DNA with relatively low levels of endotoxin. EndoFree Plasmid Kits include an integrated endotoxin-removal step to yield plasmid DNA containing <0.1 EU/g plasmid DNA.

Procedure

Following clearing of the bacterial lysate using the QIAfilter Cartridge (see "QIAfilter principle" ), the Endotoxin Removal Buffer is added to the filtered lysate which is incubated on ice. The lysate is then applied directly to a QIAGEN-tip for plasmid DNA purification.

Applications

EndoFree Plasmid Kits yield endotoxin-free DNA which is ideal for high reproducibility and efficiency in transfection (see figures "Plasmid Purification Method vs. Transfection Efficiency" and "Plasmid Purity vs. Transfection Efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and genetic vaccination and other sensitive applications.

Endotoxin levels in plasmid preparations*



*Host strain: DH5 a plasmid: pRSVcat
1 ng LPS = 1.8 EU
Calculated from data in Figure 9.19

EndoFree DNA yields high transfection efficiencies with primary cells Primary rabbit gastric parietal cells were transfected with pEGFP-N2 (CLONTECH) prepared by the methods indicated. Transfections were performed using Effectene Transfection Reagent. The data represent the percentage of cells expressing GFP as determined by scoring the number of green fluorescent cells 48 h post transfection. The transfection efficiencies represent the average from 6 to 9 replicate dishes from more than 2 different DNA preps for each purification method. (Data kindly provided by C. Chew and J. Parente, Department of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.) Plasmid Purification Method vs. Transfection Efficiency Two independent pRSVcat preparations obtained with each method shown were each transfected twice into COS-7, HeLa, and LMH cells using a liposome-mediated method and into Huh7 cells using calcium phosphate. Average transfection efficiencies are expressed as percentages relative to the efficiency obtained with DNA prepared using the QIAGEN Plasmid Kit (100%). Plasmid Purity vs. Transfection Efficiency Effect of the amount and quality of plasmid DNA on transfection efficiency in CHO SSF3 X9 cells grown in suspension under serum-free conditions. Each point represents the mean of three independent experiments; Rel. LU: relative light units. Data kindly provided by M. Zang-Gandor, Novartis AG, Basel, Switzerland.

* For purification of low-copy plasmids and cosmids, EndoFree Plasmid Mega Kits are a better choice than EndoFree Plasmid Giga Kits, due to the large culture volumes required and the limited capacity of the QIAfilter Mega-Giga Cartridge.



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