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

 Product Information

GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kits
 

Product No. Product Name Purifications
per kit
Technical Manual
Short
Protocol
NA0400S GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit 4 PDF PDF
NA0400 GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit 10 PDF PDF
NA0410 GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit 25 PDF PDF

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

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.
 

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 (Product No. L6037). The efficiency of the transfection was determined by measuring the β-galactosidase activity using the β-galactosidase Reporter Gene Activity Detection Kit (Product No. GALA-1KT).