Murakami Agnieszka M, Yonekura Manabu, Nagatomo Katsuhiro, Niwa Yasutaka, Itagaki Shirou, Murakami Manabu
Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Collaboration Center for Community and Industry, Sapporo Medical University, Sapporo, Japan.
MethodsX. 2023 Mar 30;10:102167. doi: 10.1016/j.mex.2023.102167. eCollection 2023.
DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).• strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.
DNA重组是一种用于克隆及后续功能分析的有用技术，而质粒DNA重组的标准技术一直未变。在本研究中，我们引入了一种用于质粒DNA重组的快速方法，我们将其命名为“村上系统”，以便在33小时内完成实验。为此，我们选择了以下内容：进行25个循环的PCR扩增以及使用生长迅速的菌株（培养时间为6 - 8小时）。此外，我们选择了快速质粒DNA纯化（小量制备；约10分钟）和快速限制性内切酶孵育（20分钟）。这种重组系统能够在24 - 33小时内实现快速质粒DNA重组，这在各个领域可能都有用。我们还建立了一种用于感受态细胞制备的一日方法。我们的快速重组系统允许每周进行数次质粒DNA重组，这改进了对各种基因的功能分析。
•“质粒DNA重组的快速方法（村上系统）。
•生长迅速的菌株（培养时间为6 - 8小时）。
•快速方案（PCR、电泳、DNA纯化、连接和小量制备）的组合使得质粒DNA重组能在24 - 33小时内完成。
