Endotoxin Free Plasmid Prep – Why Is It Important?

BioInnovatise Plasmid DNA Team

Updated January 29, 2024

How Does Plasmid DNA Preparation Create Endotoxins?

Plasmid prep involves the extraction and purification of plasmid DNA from bacterial cells harboring the plasmid. The goal is to obtain a pure and concentrated sample of plasmid DNA for various applications. The process of plasmid DNA preparation often results in the introduction of endotoxins into the final DNA product. These endotoxins are lipopolysaccharides (LPS) found in the outer membrane of Gram-negative bacteria, and they can be released during the lysis of bacterial cells commonly used in plasmid DNA production, as illustrated in the diagram below.

During the bacterial cell lysis step, endotoxins can be released from the bacterial cell wall, contaminating the plasmid DNA preparation. Our team is able to purify and minimize endotoxin contamination after bacterial lysis takes place. Using specialized columns, resins, and other purification techniques, all our plasmid prep productions result in a 1.8 nucleic acid 260/280 ratio, ensuring you have the high-quality plasmid DNA needed for your research.

The above diagram illustrates the plasmid prep production process at BioInnovatise which produces endotoxin free and animal component free plasmid DNA. We perform plasmid prep using our proprietary plasmid DNA preparation protocol.

What Effects Do Endotoxins Have In Downstream Plasmid DNA Applications?

Endotoxin contamination can have adverse effects in downstream applications, especially in biological assays or experiments involving mammalian cells. Having a high purity plasmid is incredibly important for your research otherwise your experimental results may not be as accurate as they should.

Having endotoxin free plasmid DNA is essential for maintaining the integrity of experimental systems, ensuring the safety of therapeutic applications, and complying with regulatory standards in various fields, including cell and gene therapy, precision medicine, and other forms of research and development. Here are some of the consequences of using plasmid DNA that does not have a high endotoxin free purity standard.

Biological Impact On Mammalian Cells: Endotoxins can trigger strong immune responses in mammalian cells. This immune response may lead to the activation of inflammatory pathways, potentially affecting cell viability, function, and overall experimental outcomes.
Cell and Gene Therapy Safety: In cell and gene therapy, where plasmid DNA is often used to deliver therapeutic genes into patient cells the presence of endotoxins can pose serious safety concerns. Our plasmid DNA team only produces research grade plasmid DNA and although our facility does not produce cGMP grade plasmid DNA, our colleagues at Maxim Biomedical do. Contact their team for more information.
Precision Medicine Accuracy: Precision medicine relies on accurate and reliable data for personalized treatments. Contamination with endotoxins in research materials can introduce variables and confounding factors that may affect the interpretation of experimental results. Ensuring endotoxin free plasmid DNA is essential for generating trustworthy data in precision medicine research.
Consistency in Research Results: In any form of research and development, maintaining consistency and reproducibility of results is paramount. Contaminated plasmid DNA can introduce variability, making it challenging to reproduce experiments or compare results across different studies. Eliminating endotoxin contamination contributes to the reliability of research findings.

What Is The 260/280 Nucleic Acid Ratio?

The 260/280 ratio is a measure of the purity of nucleic acid samples, which includes plasmid DNA. The ratio is calculated by comparing the absorbance of the sample at wavelengths 260 nm and 280 nm. A 1.8 ratio for the 260/280 absorbance is considered ideal for pure, high quality plasmid DNA, and here’s why:

Purity Assessment: The 260/280 ratio is used to assess the contamination of nucleic acid samples with proteins. A ratio of 1.8 indicates that the sample is relatively free from protein contamination. A higher ratio suggests that the sample is purer in terms of nucleic acids.
Protein Absorption at 280 nm: Proteins absorb light at 280 nm, and nucleic acids absorb light at 260 nm. A 1.8 ratio indicates that the absorbance at 260 nm is significantly higher than at 280 nm, suggesting that the contribution of protein contamination to the absorbance at 260 nm is minimal.
Optimal for Downstream Applications: In many downstream applications, such as PCR, DNA sequencing, and transfection, having high purity plasmid DNA is crucial. Contaminants like proteins or RNA can interfere with these applications, affecting their success and reliability. A 260/280 ratio of 1.8 ensures that the plasmid DNA is suitable for these downstream applications.
Consistency and Reproducibility: Maintaining a consistent 260/280 ratio across different plasmid DNA applications ensures reproducibility of results.

All of our plasmid midi prep, plasmid maxi prep, and large scale productions up to 1 gram have a 1.8 nucleic acid 260/280 ratio.

How Can I Start A Plasmid Prep Production?

Let’s get started! Our plasmid team is excited to bring your project to life. In order to get started please provide following when requesting a production:

Plasmid copy number
Specification of antibiotic resistance of the plasmid
1 μg of wildtype plasmid

Precision medicine research and development progresses everyday, and with it, the need for high quality plasmid DNA.

Want to learn more about the latest in plasmid research? Our colleagues at Science Direct and the American Society for Biochemistry and Molecular Biology are always collecting and publishing the latest information and research.

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info@bioinnovatise.com