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Mixed protease system, kit containing mixed protease system and use method of kit

A technology of mixing protease and protease, which is applied in the field of molecular biology, can solve the problems that the function has yet to be discovered, and achieve the effects of improving accuracy, ensuring fidelity, and reducing the probability of mutation

Inactive Publication Date: 2015-05-20
苏州神洲基因有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the role of extracellular PCNA in the DNA homologous recombination reaction remains to be discovered

Method used

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  • Mixed protease system, kit containing mixed protease system and use method of kit
  • Mixed protease system, kit containing mixed protease system and use method of kit
  • Mixed protease system, kit containing mixed protease system and use method of kit

Examples

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Embodiment 1

[0051] According to the method described in Example 1, suitable PCR primers were designed to clone the Kanamycin gene with a length of approximately 1.2 kb from the plasmid pDONR222 into the pUC19 vector.

[0052] 1. Preparation of vector pUC19-linearization treatment

[0053] (1) Enzyme digestion method: Two digestion sites of EcoRI and SbfI were selected to double digestion of vector pUC19, and the digested vector was tapped and purified.

[0054] (2) PCR method: Select EcoRI and SbfI two restriction sites, design forward and reverse primers, and use high-fidelity Pfu DNA polymerase for amplification; in order to avoid the impact of template plasmid DNA on subsequent experiments, PCR amplification The increased linearized vector is purified by tapping to remove the circular template plasmid and increase the positive rate.

[0055] 2. Preparation of target insert Kan

[0056] Design primers for PCR amplification of the target fragment. The primer design must ensure that the target fra...

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Abstract

The invention relates to a mixed protease system used for gene cloning and assembling, a kit containing the mixed protease system and a use method of the kit. By utilizing the mixed protease system, specificity recombinant reaction of two or more DNA can be completed at the high temperature of 40-70 DEG C. The invention also relates to the kit containing the mixed protease system. Compared with enzyme or enzyme system with the same function, the mixed protease system keeps high activity at the high temperature of 40-70 DEG C, then the specificity of recombinant reaction is improved, generation of meaningless mutation is effectively avoided, high fidelity cloning and assembling of two or more DNA can be realized, the stability is good, the reaction precision is high, and operation is simple, convenient and efficient.

Description

Technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a mixed protease system, a kit containing the protease system and a method of use thereof, and in particular to a mixed protease system for gene cloning and assembly, and a hybrid protease system containing the mixed protease system Kits and methods for gene cloning and assembly. Background technique [0002] Recombinant DNA (Recombinant DNA) technology refers to the artificial transformation and recombination of DNA molecules from different sources according to people’s needs to form chimeric DNA molecules (Chimeric DNA Molecules), which are then introduced into recipient cells to recombine Genes are amplified and expressed in the recipient cells, so that the recipient cells acquire new genetic characteristics and produce new gene products required by humans. For example, in the field of synthetic biology, the method of using combinatorial promoters can regulate an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N9/12C12N15/66
CPCC12N15/63
Inventor 王德明
Owner 苏州神洲基因有限公司
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