DNA & RNA Purification

GenElute™ HP Endotoxin-Free Plasmid Megaprep Kit

Description: The GenElute™ HP Endotoxin-Free Plasmid Megaprep Kit offers a simple, rapid, cost-effective method of isolating endotoxin-free plasmid DNA in 90 minutes or less. GenElute HP Endotoxin-Free Plasmid Kits include a filter 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 5 mgs of high-quality, endotoxin-free (≤0.1EU/µg) plasmid DNA in 90 minutes or less
  • Convenient – vacuum format and no ethanol precipitation required

 

Procedure: An overnight recombinant E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis. The lysate is cleared by filtration followed by the addition of a binding solution that has been optimized for endotoxin-free plasmid preparations. The plasmid DNA is then captured onto a silica membrane in the presence of high salts, while endotoxins are prevented from adsorbing to the membrane. Contaminants are removed by two wash steps. The bound DNA is then eluted in endotoxin-free 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).


Prepare binding column.
 

 

Resuspend overnight culture and lyse cells.
 

 

Neutralize and filter lysate.
 

 

 

Load lysate onto binding column and wash.
 

 

 

Elute plasmid DNA.
 

 

Beneficial Features
Yield

Up to 5 mg of high-copy plasmid DNA

Speed

From harvested bacterial culture to pure plasmid DNA in 90 minutes or less

Flexibility

Convenient vacuum format does not use gravity flow columns

Economic

Lower cost plus higher yields per prep equals double the savings

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Order Information
GenElute HP Endotoxin-Free Plasmid Megaprep Kits
Product Code Product Name Purifications
per kit
Technical Manual .pdf
NA0600 GenElute HP Endotoxin-Free Plasmid Megaprep Kit 5 X

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Sample Data

Table 1.


Purification Kit Plasmid Yield (mg) Endotoxin Levels (EU/mg) Time/Prep (minutes)
GenElute HP Endotoxin-Free Megaprep Kit 6.0 1-10 75
Supplier Q Endotoxin-Free Plasmid Mega Kit 2.5 1-45 210
Supplier B Plasmid Mega EF Kit 2.0 39-46 210
Supplier M High Purity Plasmid Megaprep System 1.7 13-38 90

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Figure 1. Comparison of transfection efficiencies of high copy plasmid DNA pCMV-SPORT-β-gal/DH5α into endotoxin sensitive cell line HuH-7 cells and non-endotoxin sensitive cell line CHOK-1 cells. The data shows the average and standard deviations of three replicates for both cell lines evaluated. Untransfected HuH-7 and CHOK-1 cells represent cells treated with transfection reagents without any plasmid DNA.


Graph for Transfection Efficiencies in HuH-7 Cells

 

Graph for Transfection Efficiencies in CHOK-1 Cells

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Figure 2. Maximum plasmid DNA yield obtained with different constructs using GenElute™ HP Endotoxin-Free Plasmid Megaprep Kit.


Maximum plasmid DNA yield obtained with different constructs using GenElute™ HP Endotoxin-Free Plasmid Megprep Kit.

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