{"title":"PCR产物在gateway相容质粒载体上的重叠延伸克隆。","authors":"Omid Zare-Mehrjerdi, Gracie Trader, Viktor Kirik","doi":"10.2144/btn-2023-0001","DOIUrl":null,"url":null,"abstract":"A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"75 1","pages":"363-370"},"PeriodicalIF":2.2000,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/61/ea/btn-75-363.PMC10388215.pdf","citationCount":"0","resultStr":"{\"title\":\"Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector.\",\"authors\":\"Omid Zare-Mehrjerdi, Gracie Trader, Viktor Kirik\",\"doi\":\"10.2144/btn-2023-0001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.\",\"PeriodicalId\":8945,\"journal\":{\"name\":\"BioTechniques\",\"volume\":\"75 1\",\"pages\":\"363-370\"},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2023-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/61/ea/btn-75-363.PMC10388215.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"BioTechniques\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.2144/btn-2023-0001\",\"RegionNum\":4,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioTechniques","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.2144/btn-2023-0001","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector.
A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.
期刊介绍：
BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies.
Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers:
Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions
Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices
Surveys of technical approaches related to broad fields of research
Reviews discussing advancements in techniques and methods related to broad fields of research
Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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