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

Abstract

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Developmental biology
• Model Organisms
• Molecular Biology