In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly.
In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly.
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DOI：
10.1016/j.xpro.2022.101458
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发表时间：
2022-09-16
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用于原位杂交和基因敲低的单链核糖探针或双链rna的合成通常使用需要耗时的质粒限制性酶切和低效的凝胶纯化的载体。在这里，我们提出了一种更快的方案，用于同时合成核探针和双链RNA的质粒限制性酶切和吉布森载体组装，分别用于原位和RNA干扰实验。我们用原位涡虫和RNAi分析说明了该方案，但它适用于任何生物体。从同一质粒合成正义和反义核糖探针和抑制RNA的方法，以原虫为例，但适用于任何生物体。出版者注：开展任何实验方案都需要遵守当地机构的实验室安全和伦理指导方针。用于原位杂交和基因敲低的单链核糖探针或双链rna的合成通常使用需要耗时的质粒限制性酶切和低效的凝胶纯化的载体。在这里，我们提出了一种更快的方案，用于同时合成核探针和双链RNA的质粒限制性酶切和吉布森载体组装，分别用于原位和RNA干扰实验。我们用原位涡虫和RNAi分析说明了该方案，但它适用于任何生物体。
The synthesis of single-stranded riboprobes or double-stranded RNAs for in situ hybridization and gene knockdowns often use vectors that require time-consuming plasmid restriction digests and inefficient gel purifications. Here, we present a faster protocol for the simultaneous plasmid restriction digestion and Gibson assembly of vectors for the synthesis of both riboprobes and double-stranded RNAs for in situ and RNA interference experiments, respectively. We illustrate the protocol with planaria in situ and RNAi assays, but it is applicable to any organism. Simultaneous digestion and assembly of stable vectors with Gibson assembly Synthesis of sense and antisense riboprobes and inhibitory RNA from same plasmid Protocol illustrated with planaria but applicable to any organism Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The synthesis of single-stranded riboprobes or double-stranded RNAs for in situ hybridization and gene knockdowns often use vectors that require time-consuming plasmid restriction digests and inefficient gel purifications. Here, we present a faster protocol for the simultaneous plasmid restriction digestion and Gibson assembly of vectors for the synthesis of both riboprobes and double-stranded RNAs for in situ and RNA interference experiments, respectively. We illustrate the protocol with planaria in situ and RNAi assays, but it is applicable to any organism.