DNA Ligation(vector and insert)
Ligation
The next step is the ligation of the insert into the linearized vector.
This involves the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl residues, which can be catalyzed by two different ligases: E. coli DNA ligase and bacteriophage T4 DNA ligase. The latter is the preferred enzyme because it can also join blunt-ended DNA fragments.
The efficiency of the ligation reaction depends on:
· the absolute DNA concentration. The concentration should be high enough to ensure that intermolecular ligation is favored over self-ligation but not so high as to cause extensive formation of oligomeric molecules.
For pET vectors, good results are obtained at a vector DNA concentration or approx. 1 nM (i.e. 50-100 ng vector DNA per 20-ml ligation mix).
· the ratio between vector and insert DNA. The maximum yield of the right recombinants is usually obtained using a molar ratio of insert to vector DNA of approx. 2. If the concentration of insert DNA is substantially lower than that of the vector, the ligation efficiency becomes very low.
In practise, we set-up ligation reactions with a molar ratio of insert to vector DNA of 1:1, 2:1, and 3:1.
· the cloning strategy. Higher yields of the right recombinant are obtained when the vector and insert have been prepared using two restriction enzymes and the digested vector has been gel-purified before the ligation reaction.
The ligation of blunt-ended fragments is less effective than that of sticky-ended ones. Blunt-end ligation may be enhanced by:
· high concentrations of blunt-ended DNA fragments.
· a high concentration of ligase
· a low concentration of ATP the addition of PEG 4000
Sources:
Content:
https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/restriction_enzymes/
Image:
https://www.accessexcellence.org/RC/VL/GG/inserting.php