Preparation method and application of rapid DNA ligase
A technology of DNA ligase and enzyme activity, which is applied in the field of genetic engineering, can solve the problems of low connection efficiency, time-consuming and labor-intensive gene cloning, and low DNA connection efficiency, and achieve the effect of shortening time and high gene cloning efficiency
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[0029] Example 1 Preparation of Rapid DNA Ligase from Psychrophilic Bacteria Colwellia psychrerythraea
[0030] The first step is to design and synthesize the DNA ligase gene of the psychrophilic bacterium Colwellia psychrerythraea, and insert it between the NdeI and BamHI sites of the pET28 expression vector, fuse the archaeal histone and DNA ligase at the C-terminus, and insert 12 Alanine, construction of a recombinant expression plasmid for fast DNA ligase from the psychrophilic bacterium Colwellia psychrerythraea. The N-terminal of the fast DNA ligase has 6 consecutive histidine affinity purification tags derived from the pET28 vector, which is used for immobilized nickel ion affinity chromatography purification.
[0031] In the second step, the fast DNA ligase of the psychrophilic bacterium Colwellia psychrerythraea was recombinantly expressed. The rapid DNA ligase recombinant expression plasmid of the psychrophilic bacterium Colwellia psychrerythraea was transformed int...
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