The Importance of Plasmid DNA Quality Controls

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relative w-full h-full object-cover" src="https://media.licdn.com/dms/image/v2/D4E12AQHJw20oEmyjPw/article-cover_image-shrink_720_1280/article-cover_image-shrink_720_1280/0/1724748091314?e=2147483647&v=beta&t=ZL97zruhztLVaVU4ECOu0-GIadPEe4hMpvfPIsnXmHM" fetchpriority="high" data-embed-id="cover-image" alt="The Importance of Plasmid DNA Quality Controls"> </div> </div>  </figure> <header> <section class="core-section-container relative my-3"> <h1 class="pulse-title text-display-md text-color-text lg:text-display-lg font-bold w-11/12">The Importance of Plasmid DNA Quality Controls</h1> <div class="ellipsis-menu absolute right-0 top-0"> <div class="collapsible-dropdown flex items-center relative hyphens-auto"> <button class="ellipsis-menu__trigger collapsible-dropdown__button btn-md btn-tertiary cursor-pointer !py-[6px] !px-1 flex items-center rounded-[50%] " aria-expanded="false" aria-label="Open menu" data-tracking-control-name="article-ssr-frontend-pulse_ellipsis-menu-trigger" 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pr-2 babybear:pr-0 base-main-card--link publisher-author-card"> <a class="base-card__full-link absolute top-0 right-0 bottom-0 left-0 p-0 z-[2]" href="https://www.linkedin.com/company/genscript-corporation" data-tracking-control-name="article-ssr-frontend-pulse_publisher-author-card" data-tracking-will-navigate> <span class="sr-only"> GenScript </span> </a> <img class="inline-block relative w-6 h-6 " data-delayed-url="https://media.licdn.com/dms/image/v2/D4E0BAQGdsaHfGZNX0Q/company-logo_100_100/company-logo_100_100/0/1730429152811/genscript_corporation_logo?e=2147483647&v=beta&t=pwP9uAyWw1MSZ3OJ0u4GackV78SNQB0YqhHQ_jCT74k" data-ghost-classes="bg-color-entity-ghost-background" data-ghost-url="https://static.licdn.com/aero-v1/sc/h/cs8pjfgyw96g44ln9r7tct85f" alt="GenScript"> <div class="base-main-card__info self-center ml-1 flex-1 relative break-words papabear:min-w-0 mamabear:min-w-0 babybear:w-full ">  <h3 class="base-main-card__title font-sans text-[18px] font-bold text-color-text overflow-hidden "> GenScript </h3> <h4 class="base-main-card__subtitle body-text text-color-text overflow-hidden "> Make People and Nature Healthier through Biotechnology </h4>  <div class="body-text text-color-text-low-emphasis base-main-card__metadata"> Published Aug 27, 2024 </div>  </div> <div class="base-main-card__ctas z-[3] self-center ml-3 babybear:ml-1 babybear:self-start"> <a class="base-main-card__cta btn-sm btn-secondary" href="https://www.linkedin.com/signup/cold-join?session_redirect=%2Fpulse%2Fimportance-plasmid-dna-quality-controls-genscript-corporation-2v6me%3Ftrk%3Dpublic_post&trk=article-ssr-frontend-pulse_publisher-author-card" data-tracking-control-name="article-ssr-frontend-pulse_publisher-author-card" data-tracking-will-navigate> + Follow </a> </div> </div> </div> </section> </header> <div data-test-id="article-content-blocks">  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>DNA plasmids are small, circular, double-stranded DNA molecules that exist independently of chromosomal DNA in bacterial cells and some eukaryotic organisms. They carry genes that can be beneficial for the host organism, such as antibiotic resistance genes. In biotechnology and molecular biology, plasmids are engineered to carry specific genes for various applications, including gene cloning, protein expression, and gene therapy and much more.</span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <h3><span class>The Importance of Quality Control in Plasmid DNA Preparation</span></h3> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>Ensuring the quality of plasmid DNA is critical for successful downstream applications. Below, we delve into the key quality control metrics, their significance, their impact on research, and the methodologies used for testing.</span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class="font-[700]">1. Sequencing</span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class> <ul> <li><span class="font-[700]">Description and Importance:</span><span class> Sequencing confirms the presence and correctness of the desired insert and the plasmid backbone. It ensures that no mutations or unwanted sequences have been introduced during cloning.</span></li> <li><span class="font-[700]">Impact:</span><span class> Incorrect sequences can lead to the expression of faulty or non-functional proteins, which can invalidate experimental results and lead to misleading conclusions.</span></li> <li><span class="font-[700]">How it’s tested:</span><span class> Sanger sequencing, Next-generation sequencing (NGS), Nanopore sequencing</span></li> <li><span class="font-[700]">Standard Criteria:</span><span class> 100% sequence accuracy</span></li> </ul> </span> </p> </div> <div class="inline-articles bg-color-surface-new border-hidden rounded-sm p-1 mx-auto my-1.5 mamabear:w-2/3 inline-articles__after"> <h2 class="text-xl font-medium ml-1.5 my-1 text-color-text mamabear:text-center">Recommended by LinkedIn</h2> <div class="flex flex-wrap mx-0.5 babybear:mx-1.5"> <div class="content-author-card flex flex-col max-w-[270px] h-auto rounded-md border-1 border-system-gray-30 border-solid pb-1.5 inline-article mx-auto my-1 border-hidden papabear:w-[219px] babybear:min-w-full mamabear:min-w-full h-auto bg-color-canvas-tint"> <a class="no-underline" data-tracking-control-name="inline-recommended-articles" data-tracking-will-navigate href="https://www.linkedin.com/pulse/oligonucleotide-synthesis-methods-technologies-luke-mclaughlin-uueoc"> <img class="content-image w-full h-[150px] bg-cover rounded-t-md object-cover" data-delayed-url="https://media.licdn.com/dms/image/v2/D5612AQHXFpamUHwqjA/article-cover_image-shrink_720_1280/article-cover_image-shrink_720_1280/0/1729524842830?e=2147483647&v=beta&t=nexeGxrfOF1H_96kADczEP-fxejW1AN0-kgvatg3ja4"> <div class="inline-article-title text-md text-color-text font-bold pl-2 pt-1 pr-1.5">Oligonucleotide Synthesis: Methods and Technologies</div> <div class="inline-article-meta px-2 mt-1 font-sans text-xs font-normal text-color-text-low-emphasis h-full"> <span class="inline-article-author pt-0.5"> Luke McLaughlin </span> <span class="before:middot pt-0.5 inline-article-published-date"> 7 months ago </span> </div> </a>  </div> <div class="content-author-card flex flex-col max-w-[270px] h-auto rounded-md border-1 border-system-gray-30 border-solid pb-1.5 inline-article mx-auto my-1 border-hidden papabear:w-[219px] babybear:min-w-full mamabear:min-w-full h-auto bg-color-canvas-tint"> <a class="no-underline" data-tracking-control-name="inline-recommended-articles" data-tracking-will-navigate href="https://www.linkedin.com/pulse/cloning-vectors-complete-a-z-luke-mclaughlin-epwqc"> <img class="content-image w-full h-[150px] bg-cover rounded-t-md object-cover" data-delayed-url="https://media.licdn.com/dms/image/v2/D5612AQEC-eJh2MqieQ/article-cover_image-shrink_720_1280/article-cover_image-shrink_720_1280/0/1727705559382?e=2147483647&v=beta&t=kznVGQm9l_PML3hrTk13Eb2vz8xpAKRvn7Pk2rvGKUk"> <div class="inline-article-title text-md text-color-text font-bold pl-2 pt-1 pr-1.5">Cloning Vectors: The complete A-Z</div> <div class="inline-article-meta px-2 mt-1 font-sans text-xs font-normal text-color-text-low-emphasis h-full"> <span class="inline-article-author pt-0.5"> Luke McLaughlin </span> <span class="before:middot pt-0.5 inline-article-published-date"> 1 month ago </span> </div> </a>  </div> <div class="content-author-card flex flex-col max-w-[270px] h-auto rounded-md border-1 border-system-gray-30 border-solid pb-1.5 inline-article mx-auto my-1 border-hidden papabear:w-[219px] babybear:min-w-full mamabear:min-w-full h-auto bg-color-canvas-tint"> <a class="no-underline" data-tracking-control-name="inline-recommended-articles" data-tracking-will-navigate href="https://www.linkedin.com/pulse/unlocking-intricate-tapestry-whole-genome-sequencing-tim-4kxfe"> <img class="content-image w-full h-[150px] bg-cover rounded-t-md object-cover" data-delayed-url="https://media.licdn.com/dms/image/v2/D4E12AQFBYjvDu-_eIA/article-cover_image-shrink_720_1280/article-cover_image-shrink_720_1280/0/1711803032464?e=2147483647&v=beta&t=0N82aAYJk-n2iZX8jV2VSA43-94pPp3Ov9zR6x6p8CE"> <div class="inline-article-title text-md text-color-text font-bold pl-2 pt-1 pr-1.5">Unlocking the intricate tapestry: Whole Genome…</div> <div class="inline-article-meta px-2 mt-1 font-sans text-xs font-normal text-color-text-low-emphasis h-full"> <span class="inline-article-author pt-0.5"> Tim Sandle, Ph.D., CBiol, FIScT </span> <span class="before:middot pt-0.5 inline-article-published-date"> 7 months ago </span> </div> </a>  </div> </div> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class="font-[700]">2. Restriction Enzyme Digestion</span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class> <ul> <li><span class="font-[700]">Description and Importance:</span><span class> This technique involves cutting the plasmid DNA with specific restriction enzymes that recognize and cut at particular DNA sequences. It verifies the presence, orientation, and size of the insert within the plasmid by generating fragments of predictable sizes, ensuring that the plasmid contains the correct insert.</span></li> <li><span class="font-[700]">Impact:</span><span class> Ensures that the plasmid contains the correct insert, which is crucial for successful cloning and expression experiments.</span></li> <li><span class="font-[700]">How it’s tested:</span><span class> Gel electrophoresis: After digestion, the DNA fragments are separated on an agarose gel and visualized to confirm their sizes match the expected pattern.</span></li> <li><span class="font-[700]">Standard Criteria:</span><span class> Fragment sizes: The observed fragment sizes should match the predicted sizes based on the plasmid map.</span></li> </ul> </span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class="font-[700]">3. DNA Supercoil</span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class> <ul> <li><span class="font-[700]">Description and Importance:</span><span class> Supercoiling refers to the over- or under-winding of the DNA helix, naturally occurring in plasmids as a tightly coiled conformation. Supercoiled DNA is more compact and efficient for transformation and transfection into cells.</span></li> <li><span class="font-[700]">Impact:</span><span class> Higher supercoiling improves the efficiency of plasmid uptake by cells, leading to better expression of the cloned gene.</span></li> <li><span class="font-[700]">How it’s tested:</span><span class> Gel electrophoresis: Supercoiled plasmids migrate faster than relaxed or linear plasmids, allowing their forms to be distinguished.</span></li> <li><span class="font-[700]">Standard Criteria:</span><span class> Supercoiled proportion: A high proportion of the plasmid should be in the supercoiled form (>80%).</span></li> </ul> </span> </p> </div>  <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class><a 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