Zero-background multi-fragment gene efficient assembling method

An assembly method and multi-segment technology, applied in the biological field, can solve the problems of assembly failure and reduced assembly success rate, and achieve the effect of reducing cost

Inactive Publication Date: 2019-07-30
通用生物(安徽)股份有限公司
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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 t

Method used

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  • Zero-background multi-fragment gene efficient assembling method
  • Zero-background multi-fragment gene efficient assembling method
  • Zero-background multi-fragment gene efficient assembling method

Examples

Experimental program
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Embodiment Construction

[0069] The present invention will be further elaborated below by specific examples.

[0070] 1. Strains

[0071] Host bacteria Escherichia coli TOP10.

[0072] 2. Reagents

[0073] DNA polymerase and dNTP are Anhui General Biology (System) Co., Ltd.;

[0074] Gel recovery and plasmid extraction kits are AXYGEN products;

[0075] T4 DNA ligase was purchased from Thermo Company;

[0076] T7 DNA ligase, restriction endonuclease Bsa I, EcoR I, HindIII were purchased from NEB Company;

[0077] The primers were synthesized by our company, and pUC57 and pACYC-duet were commercial vectors preserved by our company; the recombinant enzyme GenREC was developed and produced by our company.

[0078] 1. Experimental process

[0079] A zero-background efficient cloning method for gene assembly, the steps of which include:

[0080] (1) Construct the puc57-CATP carrier;

[0081] (2) construction clone has the plasmid of CATP gene fragment;

[0082] (3) Select the target gene, divide i...

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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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1027
Inventor 雍金贵
Owner 通用生物(安徽)股份有限公司
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