Cloning
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General Procedure
This should be a consensus protocol. See the bottom of this article for specific protocols.
Materials
Equipment
- gas burner
- rotating plate
- aluminium rack for PCR tubes
Media and plates
1.2- Reagents
- Other consumables
- gloves
- sterile (???) plates
- pH paper
- pasteur pipettes
- DW
Procedure
Enriching the insert
The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various purification methods and there exists several protocols for PCR
Ligation
Just see DNA ligation
Reagents
- T4 DNA ligase
- 10x T4 DNA Ligase Buffer
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)
Method
- 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
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See Bacterial transformation.
Reagents
- Competent cells: XL2-Blue (Stratagene 200150).
- Learn how to make your own by Preparing_chemically_competent_cells
- SOC medium
- ß-mercaptoethanol (ß-MAE)
Method
Screening
Either a check digest or Colony PCR will be a good way of screening your colonies. If the plasmid is of the correct size, submit for DNA sequencing
Critical steps
Troubleshooting
Notes
See Also
Cloning Software
- Digital Tools & Resources
- ApE - A Plasmid Editor (software review)
- Gene Construction Kit software review
- MCDS
Protocols
General
- Digital Tools & Resources
- Cloning and sequencing
- Cloning Checklist
- General Cloning Protocol
- Cloning Protocol
Lab-specific protocols
- Knight:TOPO TA cloning
- Wikiomics:Cloning in silico
- Wikiomics:Site Directed Mutagenesis
- Duffy:LIC Cloning
- Rutgers:DNA Ligation
- Rutgers:Transformation
- Milo:No background cloning protocol
Specific Protocols
Discussion
You can discuss this protocol.
