Preparation method of T carrier

Abstract

The invention discloses a preparation method of a T carrier, which utilizes thio-modified primers to amplify a plasmid vector; digesting the template plasmid molecule with restriction endonuclease DpnI to eliminate the negative background clone caused by the circular plasmid template; the linearized vector amplified by PCR was digested with 5'exonuclease to obtain T carrier. By the method of theinvention, the linearized fragment of the T carrier can be prepared by direct and simple PCR amplification, the technical defects of the restriction endonuclease method and the terminal deoxynucleotidyl transferase method are eliminated, and the T carrier can be obtained by simple treatment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method of a T carrier. Background technique [0002] The emergence of gene cloning technology has greatly promoted the development of modern genetic engineering and protein engineering. The traditional gene cloning technology involves the restriction endonuclease digestion of the target gene fragment and the plasmid vector, and the two essential operations of ligase ligation and circularization of the target gene and the plasmid. Due to the restriction endonuclease digestion reaction and the DNA ligase reaction, especially the restriction endonuclease digestion reaction, the efficiency directly determines whether the target gene cloning can be successful. The main disadvantages of traditional gene cloning are: 1) The selection of restriction endonucleases is cumbersome, and suitable and easy-to-use restriction enzyme sites are often not found for long-fragment ge...

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Problems solved by technology

Although this method can prepare T vectors, because terminal deoxynucleotidyl transferase can add one to several T bases at the 3' end, it is difficult to control the number of T bases added at the end, and only adding The case of a T base is the required T carrier

The removal of circular template plasmid molecules in the other two methods has no good strategy, resulting in a higher background of negative clones

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