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This should be a consensus protocol. See the bottom of this article for specific protocols.
- gas burner
- rotating plate
- aluminium rack for PCR tubes
Media and plates
- Other consumables
- sterile (???) plates
- pH paper
- pasteur pipettes
Enriching the insert
Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See Restriction digest.
The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see DNA ligation.
- T4 DNA ligase
- 10x T4 DNA Ligase Buffer
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)
- Mix reagents for a 10 μL reaction.
- Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
- Denature ligase at 65°C for 10 minutes.
- Store at -20°C.
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See Bacterial transformation.
- Competent cells: XL2-Blue (Stratagene 200150).
- Learn how to make your own by Preparing_chemically_competent_cells
- SOC medium
- ß-mercaptoethanol (ß-MAE)
- Duffy:LIC Cloning
- Knight:TOPO TA cloning
- Milo:No background cloning protocol
- Rutgers:DNA Ligation
- Wikiomics:Cloning in silico
- Wikiomics:Site Directed Mutagenesis
You can discuss this protocol.