Ligation of DNA
In order to construct new DNA molecules, DNA must first be digested using restriction endonucleases (see Restriction endonuclease digestion of DNA). The individual components of the desired DNA molecule are purified and then combined and treated with DNA ligase. The products of the ligation mixture are introduced into competent E. coli cells and transformants are identified by appropriate genetic selection. Appropriate control ligations should also be performed (see Preparation of competent E. coli and Transformation of competent E. coli).
Removal of 5' phosphates from linearized vector DNA can help prevent vector self-ligation and improve ligation efficiency. To remove 5' phosphates from DNA, add calf intestinal phosphate (CIP) buffer and 1 U CIP and incubate for 30–60 minutes at 37°C. Once the reaction is complete, inactivate CIP by heating to 75°C for 15 minutes.
- A typical ligation reaction is set up as per the table Typical ligation reaction.
- Incubate for 1–24 h at 15°C. Tip: Simple ligations with two fragments having 4 bp 3' or 5' overhanging ends require much less ligase than more complex ligations or blunt-end ligations. The quality of the DNA will also affect the amount of ligase needed.
Tip: Ligation of sticky-ends is usually carried out at 12–15°C to maintain a balance between annealing of the ends and the activity of the enzyme. Higher temperatures make annealing of the ends difficult, while lower temperatures diminish ligase activity.
Tip: Blunt-end ligations are usually performed at room temperature since annealing is not a factor, though the enzyme is unstable above 30°C. Blunt-end ligations require about 10–100-times more enzyme than sticky-end ligations in order to achieve an equal efficiency.
- Introduce 1–10 µl of the ligated products into competent E. coli cells and select for transformants using the genetic marker present on the vector (for further information, see Preparation of competent E. coli and Transformation of competent E. coli).
- From individual E. coli transformants, purify plasmid or phage DNAs by miniprep procedure and determine their structures by restriction mapping.
Tip: It is recommended to include two controls in every transformation experiment: A “mock” transformation without DNA and a transformation with a known amount of closed circular plasmid DNA.
Typical ligation reaction
|Component DNAs||0.1–5 µg
|10 mM ATP||1 µl
|T4 DNA ligase||20–500 U