Zero-background multi-fragment gene efficient assembling method

Abstract

The invention discloses a zero-background multi-fragment gene efficient assembling method. The method includes the following steps that a puc57-CATP carrier is established; plasmid in which CATP genefragments are cloned is established; a target gene is selected, the target gene is divided into small fragments according to the length of the gene for gene synthesis, second-class enzyme loci are added to the two ends of the gene, and the gene is connected to a cloning carrier; the small fragments comprise the established CATP gene fragments and are assembled through a Golden gate cloning reaction, T7DNA polymerase is used as ligase, an assembled product converts escherichia coli, and an LB panel with ampicillin and chloromycetin resistance is coated with the assembled product for screening.Under the condition that the carrier plasmid does not need to be treated in advance, by means of special carrier and fragment design, multiple gene fragments are efficiently assembled simultaneously at a time. The T7DNA ligase with higher fidelity is used, and the probability of correct assembly of the fragments is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a zero-background high-efficiency multi-segment gene assembly method. Background technique [0002] Gene cloning has achieved fruitful results in agriculture, animal husbandry and medicine in recent years. The basic principle is to connect foreign genes with cloning vectors in vitro, then transform host cells, and screen out transformants containing recombinant plasmids of target genes. [0003] Traditional gene cloning steps include: select target gene and design corresponding primer; Amplify target gene fragment with primer PCR; Select suitable (resistance marker, restriction site etc.) cloning vector (for fidelity amplification), and The PCR fragment is ligated into the cloning vector (generally, the PCR product with Taq enzyme will have an A at the end, which can be ligated with a linear vector with a T at both ends under the action of Solution 1). [0004] With the development ...

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Problems solved by technology

These subsequent background interferences will be gradually enriched during the replication of E. coli, occupying the vast majority of the total number of clones on the plate, resulting in a significant reduction in the success rate of assembly, or even failure of assembly

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