Endotoxins in purified plasmid DNA have long been believed to have a detrimental effect on transfection efficiency and viability in all cell lines, leading to the common practice of using expensive "endotoxin-free" plasmid purification systems with a threshold of less than 0.1 EU/µg. However, recent research has shown that only high levels of endotoxin, exceeding 10 EU/µg, have a negative impact on transfection, protein expression, and viability in standard cell lines [1]. It is important to note that sensitive applications, such as transfection of primary or stem cells, as well as other in vivo applications, require the use of endotoxin-free plasmid DNA with a threshold below
0.1 EU/µg.

For most transfection applications, anion exchange plasmid purification systems like the PureLink HiPure and PureLink Fast Low Endotoxin kits are highly suitable, as they produce low levels of endotoxin ranging from 0.1 to 10 EU/µg. These levels are considered excellent for most transfection purposes. However, for the most sensitive applications, it is recommended to use advanced transfection-grade endotoxin-free plasmid DNA with a threshold below 0.1 EU/µg. This helps ensure a high level of plasmid purity and helps minimize any potential negative effects on transfection efficiency and viability.

What are endotoxins?

Endotoxins are lipopolysaccharides found in the outer membrane of Gram-negative bacteria. These molecules can cause immune responses and inflammation in mammals. Endotoxins can be problematic in scientific research as they can interfere with cellular processes and lead to unreliable experimental results.

Plasmid purity specifications for downstream applications

Molecular-grade plasmid isolation technologies are generally faster, more cost-effective and are well suited for robust applications such as cloning, nucleic acid labeling, PCR, and sequencing, where high purity levels are not required. Our GeneJET plasmid DNA purification kits are well suited for molecular-grade plasmid DNA isolation.

Transfection-grade plasmid purification technologies may be used for applications that demand greater purity and higher yields than molecular-grade plasmid DNA. Our PureLink HiPure plasmid purification kits and PureLink Fast Low Endotoxin plasmid purification kits produce the higher yields and lower endotoxin levels required for more sensitive applications, such as transfection of standard cell lines and in vitro transcription. Transfection-grade plasmid DNA is also suitable for all molecular-grade applications, such as cloning or sequencing.

Advanced transfection-grade plasmid isolation technology is suited for the most sensitive applications but can also be used for all molecular and transfection-grade applications. Our PureLink Expi Endotoxin-Free plasmid purification kits offer rapid purification of large-scale, endotoxin-free (<0.1 EU/µg) plasmid that is exceptional for transfection of sensitive cell lines (i.e., primary cells) and experiments in vivo (research on gene therapy, microinjections, and vaccines).

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Downstream application		Purity grade
MolecularTransfectionAdvanced transfection
Sensitive transfection (i.e., primary cells)   
Vaccine research   
Microinjection   
Gene therapy research   
In vitro transcription   
Standard transfection   
Cloning   
Nucleic acid labeling   
PCR   
Sequencing   
Transformation   


How do endotoxins affect biological applications?

Endotoxins can significantly impact biological applications by triggering immune responses and inflammation in cells. This can lead to altered gene expression, cell death, and compromised experimental results. Therefore, it is crucial to understand how endotoxins may affect downstream applications and what level of plasmid purity is necessary to help ensure that plasmid DNA used in research is purified correctly to obtain accurate and reliable data.


How are endotoxins removed during plasmid purification?

To achieve endotoxin-free plasmid preparations, specific purification methods are employed. These methods often involve the use of chromatography techniques, such as anion exchange or affinity chromatography, which selectively remove endotoxins from the plasmid DNA solution. Additionally, endotoxin removal kits and specialized columns are available to facilitate the purification process and help ensure high-quality, endotoxin-free plasmid DNA.


How are endotoxins measured?

The quantification of endotoxins is typically performed using the Limulus Amoebocyte Lysate (LAL) assay, which relies on the clotting reaction of horseshoe crab blood cells in the presence of endotoxins. This assay provides a sensitive and accurate measurement of endotoxin levels in plasmid DNA preparations, allowing researchers to determine the purity and safety of their samples.

Read more plasmid purity FAQs


References
  1. Butash, KA et al. (2000) Reexamination of the Effect of Endotoxin on Cell Proliferation and Transfection Efficiency. Biotechniques 29(3): 610-614, 616, 618-619. 
Resources
  • Protocol Videos—Videos to help you isolate nucleic acids using a variety of techniques.
  • DNA & RNA Selection Guide—Find the right purification products.
  • Videos—View our library of DNA & RNA purification and analysis videos.
  • Application Notes—Explore our application notes from scientists sharing data for isolation products
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