DNA & RNA Purification

Endotoxin-Free Plasmid Kits


Description: Endotoxins (also known as lipopolysaccharides or LPS) are often co-purified with plasmid DNA and can significantly reduce transfection efficiencies in endotoxin-sensitive cell lines. The GenElute™ Endotoxin-free Plasmid Kits offer a simple, rapid, cost-effective method for purifying plasmid DNA with £ 0.1 EU/µg DNA for high-efficiency transfection.

  • Fast – 12 preps in less than 2 hours
  • Convenient – No lengthy gravity flow ultracentrifugation or alcohol precipitation
  • Cost effective – more preps for the money

 

Procedure: Recombinant E. coli is harvested from an overnight culture by centrifugation and subjected to a modified alkaline-SDS lysis procedure to produce a cleared lysate. Endotoxins are removed from the cleared lysate with simple extraction-phase separation steps. Plasmid DNA is further purified by adsorption onto silica in the presence of high salts. After a simple spin-wash step, the bound plasmid DNA is eluted in endotoxin-free water. The kits are operationally identical to the standard plasmid prep kits with the exception that an endotoxin removal step is inserted between preparation of the cleared lysate and DNA capture.

Application: The DNA is ready for immediate use in downstream applications such as transfection (Figure 1), restriction endonuclease digestion, cloning, sequencing and PCR amplification.


GenElute Endotoxin-free Flow Chart

Beneficial Features
Culture Volume

5 - 40 ml (Midiprep) and 25 - 130 ml (Maxiprep)

Yield

Up to 250 µg (Midiprep) and 1.2 mg (Maxiprep)

Speed

Up to 12 preps in less than 2 hours

Versatile

Bind/wash/elute method eliminates need for tedious gravity flow, or magnetic bead separations and devices

Specific

High yields with no detectable genomic DNA or RNA contamination

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Order Information
Product Code Culture Volume (ml) Plasmid
Yield (µg)
Time
Required
Number
of Preps
Technical Manual .pdf Short protocols .pdf
PLED35 5 to 40 up to 250 Hands On: < 15 min.
Total: < 95 min.
35 X X
PLEX15 25 to 130 up to 1200 Hands On: < 20 min.
Total: < 105 min.
15 X X

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Sample Data

Preparation time for different Endotoxin-free plasmid purification kits.

Figure 1. Comparison of time required per isolation using different Endotoxin-free plasmid isolation systems. Plasmid DNA was isolated following each manufacturers recommended protocol. Each system was tested multiple times and the average preparation time is shown here.   Endotoxin-free time per isolation

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Transfection of cells with plasmid isolated with different endotoxin removal kits.

Figure 2. Comparison of transfection efficiencies of plasmids prepared with different Endotoxin-free isolation methods. The data shows the average of six replicates for each named isolation method. All transfections were in CHO-K1 cells. The OD measurements were taken at 420 nm and the units of b-gal/plate were found using the Sigma b-Galactosidase Reporter Gene Activity Detection Kit. The cells were grown to ~60-75% confluency and transfected using 3 µg of plasmid DNA/15 µg of Escort IV. b-Gal activity was determined ~60-70 hours post-transfection. Error bars are the standard deviation of the 6 replicate wells tested.

Note: Plasmids prepared with Sigma's Endotoxin-free kits gave the highest transfection efficiencies.

 

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