Fung Vincent, Tiwade Palas Balakdas, Fenton Owen S
Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
STAR Protoc. 2025 Aug 6;6(3):104025. doi: 10.1016/j.xpro.2025.104025.
Traditional plasmid assembly often involves expensive or restrictive enzymatic processes. To overcome these limitations, we introduce the CloneFast guide, a streamlined approach for efficient plasmid construction. Initially, this guide advises users through bioinformatic plasmid and oligonucleotide design in silico. Subsequently, we outline practical experimental steps, including the introduction of phosphorothioate modifications via reusable modified oligonucleotides (CompetePCR), generation of precise sticky ends through a rapid iodine-mediated cleavage, and seamless fragment assembly into plasmids. CloneFast enables the completion of plasmid assembly within 3 days, offering versatility and accessibility to a wide range of research groups.
传统的质粒组装通常涉及昂贵或受限的酶促过程。为克服这些限制，我们引入了CloneFast指南，这是一种用于高效质粒构建的简化方法。首先，该指南通过生物信息学在计算机上进行质粒和寡核苷酸设计来为用户提供建议。随后，我们概述了实际的实验步骤，包括通过可重复使用的修饰寡核苷酸（CompetePCR）引入硫代磷酸酯修饰、通过快速碘介导的切割产生精确的粘性末端以及将片段无缝组装到质粒中。CloneFast能够在3天内完成质粒组装，为广泛的研究团队提供了通用性和可及性。
