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

Reagents

• LB (Luria-Bertami) agar plates
• IPTG stock solution (100 nM)
• SOC medium
• DYT medium

• 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

Restriction Digest

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.

Ligation

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.

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

1. Mix reagents for a 10 μL reaction.
2. Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
3. Denature ligase at 65°C for 10 minutes.
4. 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

See Also

Cloning Software

• Digital Tools & Resources
• ApE - A Plasmid Editor (software review)
• Gene Construction Kit software review
• MCDS

• Genome Compiler: watch these short tutorial videos to learn how to use the Cloning Wizards (simulate digest and ligate with restriction enzymes or by PCR).

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

Online Courses

Restriction Ligation cloning method - a practical guide for this cloning technique.

Discussion

You can discuss this protocol.