In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly

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https://www.sciencedirect.com/science/article/pii/S2666166722003380

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https://doi.org/10.1016/j.xpro.2022.101458
 http://hdl.handle.net/11603/31859

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UMBC Faculty Collection
UMBC Biological Sciences Department

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Author/Creator ORCID

https://orcid.org/0000-0002-9677-219X
 https://orcid.org/0000-0003-4103-6926
 https://orcid.org/0000-0003-4666-6118

Date

2022-06-14

Type of Work

journal articles
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Citation of Original Publication

Wolff, Andrew, Cynthia Wagner, Julia Wolf, and Daniel Lobo. “In Situ Probe and Inhibitory RNA Synthesis Using Streamlined Gene Cloning with Gibson Assembly.” STAR Protocols 3, no. 3 (September 16, 2022): 101458. https://doi.org/10.1016/j.xpro.2022.101458.

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Creative Commons Attribution 4.0 International (CC BY 4.0)

Subjects

Developmental biology
Model Organisms
Molecular Biology

Abstract

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.