What is plasmid purification?

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.


Why plasmid purification matters

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.


The key steps in traditional, manual plasmid purification typically include:

Traditional plasmid DNA purification
  • 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


Understanding plasmid purification

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


Preparation sizes for pDNA isolation

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

MiniprepMidiprepMaxiprepMegaprepGigaprep
Overnight bacterial culture volume1–5 mL25–100 mL100–500 mL500 mL–2.5 L2.5–5 L
Approximate yieldUp to 40 µgUp to 400 µgup to 1.5 mgUp to 5 mgUp to 15 mg
Table 1. Overnight bacterial culture volume for each plasmid prep size from miniprep (1–5 mL) to gigaprep (2.5–5 L). For each prep size, the table highlights approximate yield for pDNA.


Purity grade and endotoxin level for plasmid DNA isolation

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.

Purity gradeMolecularTransfectionAdvanced transfection
Endotoxin level>10 EU/µg0.1–1 EU/µg<0.1 EU/µg

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.

KingFisher PlasmidPro Maxi Processor

Automate your plasmid purification


Solutions for automated plasmid purification

Hands-on productivity, hands-off process

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.

 

Applied Biosystems MagMAX Pro HT NoSpin Miniprep Plasmid Purification Kit

Thermo Scientific KingFisher Plasmid Pro

image of magmax pro ht no spin kitimage of plasmid pro instrument
Prep sizes availableMiniMaxi
Purity grade/Endotoxin levelTransfection grade (0.1–1 EU/µg)
Advance Transfection grade (<0.1 EU/µg)
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    Automate your plasmid purification with KingFisher PlasmidPro

    See how automating your plasmid DNA isolation is possible using the KingFisher PlasmidPro Maxi Processor. The newest addition to the KingFisher instrument line.


    Kits for pDNA purification

    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.

    Thermo Fisher Scientific's end-to-end solutions. This workflow shows the end-to-end solutions for protein expression and how plasmid purifications is linked to the entire process. Thermo Fisher offers solutions for each step of the process. Plasmid design can be initiated with Gene Art Plasmid Services, while plasmid production can use techniques for cloning and vector insert verification, transformation and culture growth. Plasmid purification can be done using automation with KingFisher PlasmidPro Maxi Processesor, or manually with MagMAX Pro HT NoSpin, PureLink and Expi kits. The workflow can be finished with cloning, for expression in mammalian cells, transfection, and protein expression using systems like Gibco ExpiCHO.


    Transfection-grade pDNA purification

    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.

     Invitrogen PureLink Fast Low-Endotoxin Purification KitsInvitrogen PureLink HiPure Plasmid KitsPureLink HiPure Plasmid Filter Kits
     productproductPureLink HiPure Plasmid Filter Midiprep Kit
    Prep sizes availableMidi, MaxiMini, Midi, Maxi, Mega, GigaMidiMaxi
    Purity grade/Endotoxin levelTransfection grade
    (0.1–1 EU/µg)
    Transfection grade
    (0.1–1 EU/µg)
    Transfection grade
    (0.1–1 EU/µg)


    Advanced transfection-grade pDNA purification

    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.

     Thermo Scientific GeneJET Endo-Free Plasmid Maxiprep KitInvitrogen PureLink Expi Endotoxin-Free Plasmid Purification Kits
     productproduct
    Prep sizes availableMaxiMaxi, Mega, Giga
    Purity grade/Endotoxin levelAdvanced transfection grade
    (<0.1 EU/µg)
    Advanced transfection grade
    (<0.1 EU/µg)


    Molecular-grade plasmid purification

    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.

     Invitrogen PureLink Pro Quick 96 Plasmid Purification KitInvitrogen PureLink Quick Plasmid Miniprep KitThermo Scientific GeneJET Plasmid Kits
     productproductproduct
    Prep size availableSmall-scale 96-well plate formatMiniMini, Midi, Maxi
    Purity grade/Endotoxin levelMolecular grade
    (>10 EU/µg)
    Molecular grade
    (>10 EU/µg)
    Molecular grade
    (>10 EU/µg)


    Additional buffers for plasmid isolation


    Plasmid purification how-to videos

    Automated plasmid purification: How to operate

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      How to purify advance transfection-grade plasmid DNA

      Learn how to purify advance transfection-grade pDNA with KingFisher PlasmidPro maxi processor. Plasmid Filter Maxiprep Kit. Transfection-grade pDNA is suitable for most molecular-grade applications, as well as in vitro transcription and transfection.

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        How to collect pDNA and cleanup cartridge and Instrument

        Watch a video tutorial on the cleaning and decontamination process for the KingFisher PlasmidPro instrument, including how to clean the cartridge and properly dispose of waste.

        Manual plasmid purification how-to videos

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          How to purify endotoxin-free plasmid DNA

          Watch this video to learn how to purify plasmid DNA using the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit. This video shows you how to isolate and purify high-quality, endotoxin-free plasmid DNA for sensitive applications like primary cell transfection.

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            How to purify molecular-grade plasmid DNA

            This video shows you how to purify molecular-grade plasmid DNA in 15 minutes using our GeneJET Plasmid Miniprep Kit. Molecular-grade plasmid DNA is suitable for cloning, nucleic acid labeling, PCR, DNA sequencing, and transformation.

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              How to purify plasmid DNA using our megaprep kit

              Isolate transfection-grade plasmid DNA in as little as 90 minutes with the Invitrogen PureLink HiPure Expi Plasmid Megaprep Kit. This innovative proprietary anion-exchange resin in these kits combines large capacity with fast flow rate, high resolution, and efficiency.

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                How to purify transfection grade plasmid DNA

                Learn how to purify transfection grade plasmid DNA with the Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit. Transfection grade plasmid DNA is suitable for most molecular-grade applications, as well as in vitro transcription and transfection.

                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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                For Research Use Only. Not for use in diagnostic procedures.