In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly
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 rest...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722003380 |
| _version_ | 1818202031772401664 |
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| author | Andrew Wolff Cynthia Wagner Julia Wolf Daniel Lobo |
| author_facet | Andrew Wolff Cynthia Wagner Julia Wolf Daniel Lobo |
| author_sort | Andrew Wolff |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T03:02:59Z |
| format | Article |
| id | doaj.art-adf94134ff4849f6b982db0b27b860f5 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-12T03:02:59Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-adf94134ff4849f6b982db0b27b860f52022-12-22T00:40:36ZengElsevierSTAR Protocols2666-16672022-09-0133101458In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assemblyAndrew Wolff0Cynthia Wagner1Julia Wolf2Daniel Lobo3University of Maryland, Baltimore County, Baltimore, MD 21250, USA; Corresponding authorUniversity of Maryland, Baltimore County, Baltimore, MD 21250, USAUniversity of Maryland, Baltimore County, Baltimore, MD 21250, USAUniversity of Maryland, Baltimore County, Baltimore, MD 21250, USA; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166722003380Developmental biologyModel OrganismsMolecular Biology |
| spellingShingle | Andrew Wolff Cynthia Wagner Julia Wolf Daniel Lobo In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly STAR Protocols Developmental biology Model Organisms Molecular Biology |
| title | In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly |
| title_full | In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly |
| title_fullStr | In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly |
| title_full_unstemmed | In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly |
| title_short | In situ probe and inhibitory RNA synthesis using streamlined gene cloning with Gibson assembly |
| title_sort | in situ probe and inhibitory rna synthesis using streamlined gene cloning with gibson assembly |
| topic | Developmental biology Model Organisms Molecular Biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722003380 |
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