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Gene clone plasmid based on Phi BT1 integrase and Phi C31 integrase

An integrase, plasmid technology, applied in the field of biotechnology and gene therapy, can solve problems such as difficulties

Inactive Publication Date: 2008-04-09
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It becomes very difficult to use a single phage site-specific integrase for this case

Method used

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  • Gene clone plasmid based on Phi BT1 integrase and Phi C31 integrase

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Embodiment Construction

[0030] 1. Preparation of plasmid (pBCFX+ and pBCFX-) vectors containing Phi BT1 integrase and Phi C31 integrase corresponding ATT cloning sites

[0031] (1) Preparation of plasmid backbone: reaction system (pBCPB+10ug (5ul), Sal I 5u (Bio lab.1ul), 10X Sal Ibuffer 5ul (Bio lab.), H 2 O 39ul) at 37°C for 12h, use Qiagen PCR extract kit to purify (add PBBuffer (Qiagen) 500ul to the reaction and mix well, add the mixture to PCR extract column (Qiagen) and centrifuge at 1200rpm for 1min, then centrifuge with 0.5ml PE buffer (Qiagen) at 1200rpm for 1min Wash once, then centrifuge at 1200rpm for 1min once, add 50ul Elute buffer (Qiagen) to the PCRextract column, centrifuge at 1200rpm for 1min to elute) to collect the product as the plasmid backbone.

[0032] (2) Preparation of Phi BT1-specific attP fragments: PCR system (primers (SEQ №1; SEQ №2), Pfu DNA polymerase 1u (1ul, Sagon), Pfu DNA polymerase buffer 5ul (Sagon), Phi BT genomic DNA 1ul, H2O 39ul), PCR cycle (94°C 1min, 94°C ...

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Abstract

The invention pertains to biotechnology and gene engineering fields, in particular to a gene cloning plasmid (pBCFX+ and pBCFX-) based on Phi BT1 integrase and Phi C31 integrase and the application thereof. The plasmid uses Phi BT1 integrase and tandem sequence of Phi C31fang integrase corresponding to attP site with interposition of LacZ expression frame as replacement cloning site of a plasmid vector (pBCFX+ and pBCFX-) with insertional inactivation which is used for doing blue-white screening. The cloning method based on the plasmid mainly comprises mixture of Phi BT1 integrase and Phi C31 integrase as site-directed integration kinase and additionally adding corresponding attB sequence to the two sides of inserting DNA fragment by using PCR, etc. according to the purpose to determine the direction of insertion fragment. The cloning system manipulates inserting DNA which contains special incision enzyme sites with convenience and has original predominance in manipulating Klenow fragment. The system simplifies the method of operation on recombinant DNA because of the omitting of manipulation of restricted enzyme, phosphatase and joinase and has extensive application prospect in gene engineering.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and gene therapy, and specifically relates to a plasmid (pBCFX+ and pBCFX-) based on Phi BT1 integrase and Phi C31 integrase, and a gene cloning method and application thereof. Background technique [0002] Gene manipulation is the foundation of modern biological science and biotechnology. Genetic manipulation using specific restriction sequences and homologous sequences is a widely used gene manipulation method at present. However, with the development of genetic manipulation, the above-mentioned technologies have presented a series of problems. If restriction enzyme technology is used, once a corresponding site within the gene is encountered in the process of gene manipulation, gene manipulation will become very complicated or even impossible. In addition, the laboratory is equipped with a large number of complicated endonucleases, which also affects the operating cost and efficiency of ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
Inventor 陈金中李智慧姚纪花田聆薛京伦
Owner FUDAN UNIV
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