Preparation method of cloning vector based on seamless cloning, and kit

Abstract

The invention provides a rapid cloning vector based on seamless cloning, a preparation method of the rapid cloning vector, and a kit containing the rapid cloning vector. The preparation method comprises following steps: PCR amplification is adopted to obtain target genes, wherein an obtained PCR product is characterized in that the two terminals, from outside to inside, are the homologous sequences of any Escherichia coli expression vector multiple cloning site single restriction enzyme restriction enzyme cutting site upstream / downstream 15 to 20bp, and a same restriction enzyme recognition sequence; TA cloning is adopted to construct recombinant plasmids; enzyme digestion is carried out using the same restriction enzyme, T vector large segments are recycled, T4DNA ligase is adopted for construction of pWMU-19T series vectors after intralooping. Compared with TA cloning vectors, the rapid cloning vector possesses following advantages: PCR product A adding reaction is not needed, vectorconstruction can be completed in 30min, blue-white selection of positive clone is not needed, positive clone rate is larger than 90%, and high flux construction is realized.

Description

technical field [0001] The invention relates to a cloning vector based on seamless cloning technology, a cloning method using the vector, a preparation method of the vector and a specific embodiment of a kit containing the vector, belonging to the field of genetic engineering. Background technique [0002] Cloning vectors are one of the most commonly used tools for gene preservation, amplification, sequencing, expression, and in vitro transcription and translation, and T vectors are currently the most commonly used cloning vectors for cloning PCR products. The application of this vector is based on The principle of T-A cloning technology is that during the PCR amplification process, Taq DNA polymerase has a terminal transferase activity independent of the template, which can add a protruding A base to the 3' end of the PCR amplification product, so it can be compared with The T vectors with a single T base tail at the 3' end were complementary paired and ligated to construct...

AI Technical Summary

Problems solved by technology

[0004] Although the T-A cloning method has the advantages of simple operation and high positive cloning rate compared with the clone construction method based on restriction endonuclease digestion and ligation, it also has some shortcomings, as follows: (1) For example, in order to ensure that the PCR process There will be no base mutations in the PCR product, and high-fidelity enzymes need to be used to amplify the target gene, and the high-fidelity enzymes have 3'-5' exonuclease activity, and no additional bases will be added to the 3' end of the PCR product, but need Recovering the PCR product and then using Taq enzyme to add A reaction will make the experimental operation cumbersome; (2) The preparation steps of the T vectors currently on the market are relatively cumbersome, the quality is unstable, the price is expensive, and the non-recombination background is high , cannot be reused, and is not suitable for laboratory preparation and production; (3) blue-white screening method, it is difficult to distinguish false negative colonies; (4) traditional T-A cloning method, cannot control the direction of the target gene inserted into the vector; (5) The T vectors prepared by the first method have problems such as low tailing efficiency and the sequences at both ends of the linearized plasmid are sometimes partially deleted during the tailing process; (6) The T vectors prepared by the second method have pre-T The vector may be incompletely digested and the ligation efficiency is low after digestion

And some endonucleases are expensive, or there are multiple recognition sites on common cloning vectors easily, thus limiting its application; (7) Although the T vector prepared by the third method has a very low non-recombination background, However, on the one hand, the method of PCR amplification will increase the operation steps, on the other hand, it is necessary to use expensive high-fidelity enzymes to amplify the carrier, which increases the production cost

Images