How important is the level of endotoxin for your transfections?
For the vast majority of standard laboratory cell types, even very high levels of endotoxin have virtually no effect on transfection efficiency or toxicity. “We observed that endotoxin levels greater than 2000 EU/ug DNA are required to significantly inhibit transfection for most of the cell lines tested.”
This is what the scientific literature states:
“Specifically, the transfection results using anion-exchange (PureLink® HiPure) and endotoxin-free purified DNA were indistinguishable in the cell lines tested. It may not be necessary to purify DNA using a system that contains endotoxin-free purification components and protocols because the use of a highly efficient anion-exchange, resin-based system provides sufficiently pure DNA without the labor associated with CsCl2 density gradients.”
Many companies claim that you should use ”endotoxin-free” kits for your transfection experiments!
“We observed that endotoxin levels greater than 2000 EU/ug DNA are required to significantly inhibit transfection for most of the cell lines tested.”
Purelink® HiPure Plasmid Purification Kits
|The Simple Solution for Transfection-Grade Plasmid|
|The Myth||Endotoxin contamination can decrease transfection efficiency in many of the cell lines used for transfection|
|The Scientific Reality||Invitrogen provides a midi and maxi solution for the highest yield and transfection grade purity (0.1 EU/ug) for worry-free downstream performance|
|Our Solution||The presence of endotoxins, released during cell lysis, in purified plasmid DNA can reduce transfection efficiency and subsequent protein expression levels. Starting endotoxin levels can vary in bacterial lysates, but they are significantly reduced during the purification process. Plasmid DNA purified with PureLink® HiPure Plasmid Kits have measured endotoxin levels that are equivalent to endofree kits (0.1 EU/ug) without additional workflow steps.|
Figure 1—Plasmid DNA purified using a PureLink® HiPure Plasmid Kit shows lower endotoxin levels compared to other preparation methods. To test the PureLink® Kit, TOP10 E. coli cells were transformed with pcDNA™3.1/His/lacZ plasmid and grown in LB medium. Endotoxin levels were determined by an independent testing facility using the Limulus amoebocyte lysate (LAL) qualitative gel clot method. Qiagen kit data derived from product instruction manuals. CsCl data derived from published literature.
|Cell Lines Compatible with PureLink® HiPure Kits|
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Maximizing Transfection Results
Success in transfection relies on high transfection efficiency and low cytotoxicity. Transfection efficiency is a measure of the transport of a molecule―in this case plasmid DNA—into a cell. This efficiency is influenced by many different conditions, including the quality of the plasmid DNA.
Plasmid DNA purified in a single pass with PureLink® HiPure Plasmid Kits supports high transfection efficiencies with the low cytotoxicity seen with DNA prepared with other high-purity, anion exchange-based purification kits. According to research done in 2000, cell lines used for transfection studies do not require endotoxin-free DNA. In fact, only endotoxin levels of at least 10,000 EUs significantly affect cell proliferation and viability.
PureLink® HiPure Kits Can Purify Plasmid DNA for Many Downstream Applications
Downstream applications for purified plasmid DNA include:
- Transfection, including endotoxin-sensitive cells
- In vitro transcription
- Restriction analysis, ligation, labeling, etc.
- Manual and automated sequencing, including capillary sequencing
- Gene therapy
Other Products of Interest
In addition to DNA and RNA isolation products, Life Technologies offers an expansive portfolio of solutions for analysis and quantitation of tissue and cultured cell genomic DNA.