Cloning a Gene into a Plasmid

Also called

recombinant DNA · recombinant DNA technology · gene cloning · cloning vector · plasmid · plasmid vector · pBR322 · restriction enzyme · restriction endonuclease · EcoRI · molecular scissors · sticky ends · cohesive ends · palindromic sequence · recognition site · GAATTC · DNA ligase · molecular glue · phosphodiester bond · origin of replication · ori · selectable marker · antibiotic resistance gene · transformation · competent cells · recombinant plasmid · insert orientation · screening recombinants · colony PCR · biotechnology principles and processes · tools of recombinant DNA technology

Topics

• A restriction enzyme recognises a palindromic site (5'-GAATTC-3' for EcoRI) and cuts the sugar-phosphate backbone of…
• EcoRI cuts G^AATTC on each strand, four bases apart, so every cut end carries a 5'-AATT overhang (sticky end).
• Cutting vector and insert with the same enzyme gives compatible sticky ends that can base-pair.
• Ends that do not match (for example 5'-GATC against AATT) cannot pair, so they cannot be joined.
• Paired sticky ends are held by hydrogen bonds; the backbone still has nicks until DNA ligase forms phosphodiester bonds.
• Ligation is complete only when every junction is sealed on both strands…
• With one enzyme at both ends, the insert can go in either way round, so orientation has to be checked.
• The origin of replication lets the plasmid be copied inside the host bacterium.
• Antibiotic selection keeps every cell carrying the vector, empty or recombinant…
• Screening (here, PCR across the site and an insert-to-vector PCR) separates empty from recombinant vectors and finds…

About this model

A GLB file of about 1525 KB, shown in Lably's 3D viewer and, on a phone, placed in your room with AR. Credit: Bacterial cell shell: "Bacterial cell" by Barbara Henn, Tactiles (tactiles.eu), CC BY-SA 4.0, flagellum removed and simplified. DNA helix: PDB 1BNA, Drew, Wing, Takano, Broka, Tanaka, Itakura and Dickerson (1981), PNAS 78:2179, via RCSB PDB, CC0 1.0. Plasmid, insert, sequence close-up, enzymes, cells contents, screening board, process tiles and all motion: made for Lably. | CC BY-SA 4.0. The model contains a simplified bacterial-cell shell from Tactiles (Barbara Henn, CC BY-SA 4.0), so the whole model is shared under CC BY-SA 4.0. The DNA helix uses PDB 1BNA coordinates (CC0 1.0). All other parts and all motion were made for Lably.

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