Total steps. 3.
Step 1. and 2. are involved in the activation of the 5' phosphate in the nick.
AMP group is transferred to lysine amino acid in the first residue on the enzyme and later 5' phosphate in the nick of DNA strand.
Step 3. There is attack of 3' hydoxyl group on the phosphate, which causes the displacement of the AMP. and then the phosphodiester bond seals the strand.
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ReplyDeleteNote that, originally, biochemical nomenclature distinguished synthetases and synthases. Under the original definition, synthases do not use energy from nucleoside triphosphates (such as ATP, GTP, CTP, TTP, and UTP), whereas synthetases do use nucleoside triphosphates. It is also said that a synthase is a lyase (a lyase is an enzyme that catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure) and does not require any energy, whereas a synthetase is a ligase (a ligase is an enzyme that binds two chemicals or compounds) and thus requires energy.
Enzymatics' high-concentration T4 DNA ligase in combination with the 2X Rapid Ligation buffer greatly stimulates the rate and efficiency blunt-end ligation, therefore long incubations (>10 minutes) are NOT recommended and can greatly reduce the transformation efficiency of ligation products. In order to maximize transformation efficiency of the correct insert/vector combination, the following protocol is recommended.