The digested vector, water, enzyme, and the corresponding buffer are involved in the ligation process. The ligase enzyme from prokaryotes differs from that of eukaryotes in that the cofactor required for prokaryotic ligases is ATP, while eukaryotic ligases require NADH. This enzyme forms a phosphodiester bond between the 5’ phosphate of one nucleotide and the 3’ hydroxyl of an adjacent nucleotide.
Initially, the enzyme binds to the gamma phosphate of ATP through its arginine amino acid, resulting in the release of pyrophosphate. Subsequently, the free 3’ hydroxyl of the opposite strand attacks the phosphate, leading to the separation of the phosphate from the gamma phosphate cofactor and the formation of a new phosphodiester bond with the 3’ hydroxyl. Most reactions are carried out in a volume of 10 microliters. Depending on the length of the target gene sequence and the vector sequence, specific amounts are mixed according to the formula below and incubated with the enzyme mixture for ligation.
The procedure is conducted according to the instructions for each enzyme. Most reactions are performed in a volume of 20 microliters. Based on the length of the target gene sequence and the vector sequence, specific amounts are mixed according to the ligation formula and incubated with the enzyme mixture for ligation.
Ligation is the most critical step in cloning and must be performed with precision, as it is used to insert a gene into a vector or to connect DNA fragments.