Plasmid DNA Isolation

Plasmid DNA (pDNA) isolation, also known as plasmid purification, is the process of extracting and purifying pDNA from bacterial cells, allowing scientists to obtain a highly concentrated and pure form of the plasmid for further analysis and experimentation.

Thermo Fisher Scientific offers a wide range of pDNA extraction products designed to isolate pDNA at the purity and scale you need. Choose from our selection of high-performing, cost-effective plasmid purification technologies that can help you overcome common plasmid prep issues like low recovery and the presence of impurities.

This process is critical because pDNA serves as a foundational tool in genetic engineering, enabling applications like cloning, gene expression studies, and therapeutic development. High-quality pDNA is essential for helping ensure accurate, reliable results in vital downstream projects, such as vaccine production and cell and gene therapy, helping to advance scientific breakthroughs.

• Harvest bacteria: 20–30 minutes of centrifugation
• Resuspend for lysing and neutralization: 15–20 minutes via manipulation at bench
• Clarify lysate: 5–10 minutes using centrifuge, syringe, or vacuum; “ER reduction”
• Bind, wash, and then elute precipitate: 45–60 minutes using drip technique and/or centrifugation
• Resuspend in solution: 10 minutes of manipulation at bench

Total estimated time: 95–130 minutes

There are two main considerations for plasmid purification: preparation sizes and purity grades. Both directly influence the success of downstream applications. From small-scale research to large-scale therapeutic production, these components—along with the appropriate purity/endotoxin level—help ensure the right amount of pDNA is present to achieve desired results that are both reliable and reproducible

Plasmids come in different sizes, including small (1–10 kb), medium (10–50 kb), and large (>50 kb). Small plasmids are excellent for basic cloning and gene expression studies, medium plasmids are used for more complex genetic engineering tasks, and large plasmids are suitable for creating genomic libraries and large-scale genetic projects. Understanding the appropriate media and plasmid size is crucial for achieving successful outcomes in genetic research and biotechnology.

Plasmid growth media, such as LB Broth, Terrific Broth (TB), SOC Media, and 2xYT are essential for cultivating bacterial cells that contain plasmids. These nutrient-rich media enhance plasmid yield, optimize bacterial growth conditions, and improve transformation efficiency, making them versatile tools for various molecular biology experiments.

Read more about plasmid prep sizes in our blog article:Plasmid Kit Sizes: Applications in Scientific Research

When it comes to plasmid isolation, scientists have a range of options depending on their specific needs. Molecular-grade plasmid isolation technologies offer speed and cost-effectiveness, making them excellent for robust applications like cloning, nucleic acid labeling, PCR, and sequencing, where high purity levels are not essential.

On the other hand, transfection-grade plasmid purification technologies are designed for applications that require greater purity and higher yields, such as transfection of standard cell lines and in vitro transcription. Transfection-grade plasmid DNA can also be used for molecular-grade applications. For the most sensitive applications, such as transfection of sensitive cell lines or in vivo research, advanced transfection-grade plasmid isolation technology is recommended. This technology helps ensure the highest purity and can also be used for all molecular and transfection-grade applications.

Learn more: Plasmid DNA purity grades

Robust purification of plasmid DNA, in the amount and purity required for the downstream application of interest, is the key step of any protein expression workflow. Learn how to obtain transfection-grade plasmids and use them with Gibco ExpiCHO and Expi293 transient mammalian expression systems.

Explore: ExpiCHO Expression System

Automated plasmid purification is an advanced technology that utilizes robotics to streamline the process. Thermo Fisher provides automated plasmid purification solutions that offer high-throughput and centrifuge-free purification of transfection-grade plasmid DNA. These solutions minimize human error and help ensure reproducibility by precisely controlling purification steps and reagent volumes. For mini scale applications, specialized magnetic-bead purification kits are available for efficient plasmid DNA isolation on KingFisher platforms with liquid handling capabilities. Likewise, for maxi scale automation, the KingFisher PlasmidPro maxi processer offers a hands-free experience with prefilled cartridges. The purified plasmid DNA obtained through this process can be used for a range of downstream applications, including transfection, vector preparation, enzymatic modifications, cloning, sequencing, and in vitro transcription/translation.

During the plasmid purification process, large amounts of endotoxins are released from lysed bacteria, which can negatively impact cell growth, cell viability, transfection efficiency, protein expression, and even mimic sepsis in humans. Thermo Fisher offers purification kits that span necessary pDNA purity levels, including transfection-grade, advanced transfection-grade, and molecular-grade kits.

Transfection-grade plasmid isolation kits are specifically designed for scientists who need to extract plasmid DNA (pDNA) with minimal levels of endotoxins from bacterial cultures. To address endotoxin concerns, our kits designed for low-endotoxin plasmid DNA isolation carefully remove these endotoxins, helping ensure the integrity of the isolated plasmid DNA. These kits offer a fast and easy method of plasmid purification, helping provide high purity and yield for sensitive downstream applications. Whether you are working with small or large prep sizes, our transfection-grade plasmid isolation kits are tailored to help meet your specific needs, enabling you achieve reliable and reproducible results.

For scientists who require an even higher level of endotoxin removal, we offer advanced transfection-grade plasmid isolation kits. These kits go beyond the standard transfection-grade kits and provide an enhanced level of endotoxin removal, helping ensure the utmost purity of the isolated plasmid DNA. With our advanced transfection-grade plasmid isolation kits, you can confidently perform downstream applications that demand the highest quality pDNA. These kits are excellent for researchers working on critical experiments where even trace amounts of endotoxins can have a significant impact on their results. Using our advanced-transfection grade plasmid isolation kits can help eliminate the risks associated with endotoxins and obtain pure plasmid DNA for your scientific endeavors.

For fast, easy, and reliable plasmid purification, our Invitrogen PureLink and GeneJET molecular-grade plasmid DNA (pDNA) isolation kits produce pure, ready-to-use plasmids that are compatible with a range of standard molecular biology applications. Molecular-grade kits are suitable for transformation of bacterial and yeast cells; however, they may not be well suited for transfection of sensitive mammalian cells due to the presence of endotoxins. For transfection applications, please see the transfection-grade plasmid purification kits above.

• PureLink HiPure BAC Buffer Kit
• PureLink Expi Endotoxin-Free Buffer Set