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

Andrew Wolff; Cynthia Wagner; Julia Wolf; Daniel Lobo

doi:10.1016/j.xpro.2022.101458

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

STAR Protoc. 2022 Jun 14;3(3):101458. doi: 10.1016/j.xpro.2022.101458. eCollection 2022 Sep 16.

Authors

Andrew Wolff¹, Cynthia Wagner², Julia Wolf², Daniel Lobo³

Affiliations

¹ University of Maryland, Baltimore County, Baltimore, MD 21250, USA. Electronic address: awolff@umbc.edu.
² University of Maryland, Baltimore County, Baltimore, MD 21250, USA.
³ University of Maryland, Baltimore County, Baltimore, MD 21250, USA. Electronic address: lobo@umbc.edu.

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.

Keywords: Developmental biology; Model Organisms; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Cloning, Molecular
In Situ Hybridization
Plasmids / genetics
RNA Interference
RNA, Double-Stranded* / genetics

Substances

RNA, Double-Stranded

Grants and funding

R35 GM137953/GM/NIGMS NIH HHS/United States