The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

中文翻译：

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具有通用 MCS 设计和细菌体内组装的简化质粒克隆

快速准确地将 DNA 序列克隆到质粒中的能力对于分子生物学研究至关重要。无缝克隆技术的最新发展在质粒构建方面取得了重大进展，但为了省时省力的目的，总是需要简单可靠的工具。我们开发并标准化了基于通用 MCS（多克隆位点）设计和细菌体内组装的质粒克隆方案。使用这种方法，载体首先通过 PCR（聚合酶链式反应）或限制性消化进行线性化。然后可以将少量（10~20 ng）这种线性载体与PCR扩增的插入片段（与载体的摩尔比为5倍）混合，并直接转化到感受态大肠杆菌细胞中，通过体内组装获得所需的克隆。由于我们使用 36 bp 通用 MCS 作为同源接头，因此可以将具有 ~ 15 bp 兼容末端的任何 PCR 扩增插入片段以高保真度和效率克隆到载体中。因此，消除了根据各种载体序列和以下 PCR 程序重新设计插入扩增引物的需要。我们的方案显着减少了制备转化反应的动手时间，具有出色的可靠性，并被证实是一种快速且通用的质粒克隆技术。该协议主要包含混合步骤，使其成为现代生物学研究中非常自动化且有前途的工具。无需根据各种载体序列和以下 PCR 程序重新设计插入扩增引物。我们的方案显着减少了制备转化反应的动手时间，具有出色的可靠性，并被证实是一种快速且通用的质粒克隆技术。该协议主要包含混合步骤，使其成为现代生物学研究中非常自动化且有前途的工具。无需根据各种载体序列和以下 PCR 程序重新设计插入扩增引物。我们的方案显着减少了制备转化反应的动手时间，具有出色的可靠性，并被证实是一种快速且通用的质粒克隆技术。该协议主要包含混合步骤，使其成为现代生物学研究中非常自动化且有前途的工具。