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.
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[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] ...
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[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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