DNA & RNA Purification

GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit


Description: The GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit offers a novel and efficient method of isolating endotoxin-free plasmid DNA in 40 minutes. GenElute HP Endotoxin-Free Plasmid Kits include a filter syringe for clearing lysates and binding columns that are used with a vacuum format.

  • Sophisticated – newest technology ensures improved performance
  • Novel – includes patent-pending endotoxin removal system
  • Plentiful – up to 1.2 mgs of high-quality, endotoxin-free (≤0.1EU/µg) plasmid DNA in 40 minutes
  • Convenient – vacuum format and no alcohol precipitation

 

Procedure: An overnight E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis procedure. The cleared lysate is mixed with Binding Solution, which includes reagents that inhibit endotoxins from adsorbing to the Binding Column while promoting the adsorption of plasmid DNA. No separate endotoxin removal step is required. After washing, the DNA is eluted in buffer or water.

Application: High-quality, endotoxin-free DNA is ready for immediate use for the most demanding applications including transfection with endotoxin-sensitive cells (Figure 1).




Beneficial Features
Culture Volume

150 ml

Yield

Up to 1.2 mg of high-copy plasmid DNA

Speed

From harvested bacterial culture to pure plasmid DNA in 40 minutes

Flexible

Convenient vacuum format does not use gravity flow columns

Economic

Lower cost plus higher yields per prep equals more than double the savings

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Order Information
GenElute HP Endotoxin-Free Plasmid Maxiprep Kits
Product Code Product Name Purifications
per kit
Technical Manual .pdf Short
Protocol .pdf
NA0400S GenElute HP Endotoxin-Free Plasmid Maxiprep Kit 4 X X
NA0400 GenElute HP Endotoxin-Free Plasmid Maxiprep Kit 10 X X
NA0410 GenElute HP Endotoxin-Free Plasmid Maxiprep Kit 25 X X

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Sample Data

Table 1. 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.


Purification System Plasmid Yield (mg) Endotoxin Levels (EU/µg) 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

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Transfection of pCOP-Green-C into HuH-7 cells.

Graph for Preparation Time  

Figure 1. 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 µg 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.

* pCOP-Green-C is licensed from Evrogen, JSC.

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Comparison of transfection efficiencies into HuH-7 cells.

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 β-galactosidase activity using the β-galactosidase Reporter Gene Activity Detection Kit (Sigma Product Code GALA-1KT).

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