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

A cloning vector and seamless cloning technology, applied in the field of genetic engineering, can solve the problems of incomplete enzyme cleavage, inability to control the direction of the target gene inserted into the vector, cumbersome experimental operation, etc., and achieve the effect of high recombination efficiency.

Pending Publication Date: 2018-09-04
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
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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 meth

Method used

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

Examples

Experimental program
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Example Embodiment

[0032] Example 1. A preparation method of E. coli cloning vector based on seamless cloning technology

[0033] 1. The construction of pWMU19-T pre-cloning vector

[0034] 1.1 PCR amplification of human bispecific mitogen-activated protein kinase kinase 5 (MEK5) target gene

[0035] 1.1.1 The PCR amplification MEK5 gene system and conditions are as follows:

[0036]

[0037] 95℃, 3min; 95℃, 10sec, 56.3℃, 10sec, 72℃, 60sec, 35 cycles; 72℃, 10min, 4℃ storage.

[0038] 1.1.2 Dpn I digested methylated MEK5-pCMV5 plasmid template

[0039] The digestion system and conditions are as follows:

[0040]

[0041] Incubate at 37°C for 1 hour or more.

[0042] 1.1.3 Glue recovery of digested products:

[0043] After the enzyme digestion reaction, the product was subjected to 1% low melting point agarose gel electrophoresis, and the target DNA fragment was cut under a long-wave ultraviolet lamp, and recovered with the TaKaRa gel recovery kit.

[0044] 1.1.3 A addition reaction of recovered product

[0045] ...

Example Embodiment

[0092] Example 2. An E. coli cloning vector kit based on seamless cloning technology

[0093] 1. An E. coli cloning vector kit instruction based on seamless cloning technology

[0094] 1.1 Introduction to the kit

[0095] This kit constructs the pWMU-19T series vector after transforming the commonly used TA cloning vector. The use of this series of cloning vectors is based on seamless cloning technology and has the following characteristics: the former vector can be recycled, and a linearized vector is prepared by single enzyme digestion. Both ends of the linearized vector contain 15-20bp homologous arm sequences, and PCR products do not need to be added. In the A reaction, the vector construction can be completed in 30 minutes, and clones can be constructed at high throughput without blue and white spots to screen positive clones, and the positive clone rate is greater than 90%.

[0096] 1.2 Application range

[0097] Cloning PCR products

[0098] 1.3pWMU 19-T series vector map

[0099...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66
CPCC12N15/66C12N15/70
Inventor 庞一林谭国强吕建新李江辉李唐张涛孙倩倩韩琴霞
Owner WENZHOU MEDICAL UNIV
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