Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes
Abstract Background Insertion of engineered DNA fragments into bacterial vectors is the foundation of recombinant DNA technology, yet existing methods are still laborious, require many steps, depend on specific vector configuration, or require expensive reagents. Results We have developed a method,...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-10-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-018-0359-7 |
| _version_ | 1818315761933877248 |
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| author | Matthew D. Fischer Emmanuel Mgboji Zhongchi Liu |
| author_facet | Matthew D. Fischer Emmanuel Mgboji Zhongchi Liu |
| author_sort | Matthew D. Fischer |
| collection | DOAJ |
| description | Abstract Background Insertion of engineered DNA fragments into bacterial vectors is the foundation of recombinant DNA technology, yet existing methods are still laborious, require many steps, depend on specific vector configuration, or require expensive reagents. Results We have developed a method, called “Pyrite” cloning that combines the traditional restriction enzyme digestion and ligation reaction in a single tube and uses a programmed thermocycler reaction, allowing rapid and flexible cloning in a single tube. After the Pyrite reaction and transformation, approximately 50% colonies contain the expected insert, which can be easily and quickly determined by colony PCR or blue-white colony screening. We also demonstrated that Pyrite cloning can be applied for different cloning purposes. Conclusions The Pyrite cloning method reported here is a single tube and programmed reaction cloning with restriction enzymes. Compared to other cloning methods, Pyrite cloning is flexible, inexpensive, simple, and highly efficient. |
| first_indexed | 2024-12-13T09:10:41Z |
| format | Article |
| id | doaj.art-e53fbd112c8740bbb86b3395fc0b3cc0 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-13T09:10:41Z |
| publishDate | 2018-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-e53fbd112c8740bbb86b3395fc0b3cc02022-12-21T23:52:56ZengBMCPlant Methods1746-48112018-10-011411810.1186/s13007-018-0359-7Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymesMatthew D. Fischer0Emmanuel Mgboji1Zhongchi Liu2Department of Cell Biology and Molecular Genetics, University of MarylandDepartment of Cell Biology and Molecular Genetics, University of MarylandDepartment of Cell Biology and Molecular Genetics, University of MarylandAbstract Background Insertion of engineered DNA fragments into bacterial vectors is the foundation of recombinant DNA technology, yet existing methods are still laborious, require many steps, depend on specific vector configuration, or require expensive reagents. Results We have developed a method, called “Pyrite” cloning that combines the traditional restriction enzyme digestion and ligation reaction in a single tube and uses a programmed thermocycler reaction, allowing rapid and flexible cloning in a single tube. After the Pyrite reaction and transformation, approximately 50% colonies contain the expected insert, which can be easily and quickly determined by colony PCR or blue-white colony screening. We also demonstrated that Pyrite cloning can be applied for different cloning purposes. Conclusions The Pyrite cloning method reported here is a single tube and programmed reaction cloning with restriction enzymes. Compared to other cloning methods, Pyrite cloning is flexible, inexpensive, simple, and highly efficient.http://link.springer.com/article/10.1186/s13007-018-0359-7PyriteCloningRestriction digestionSingle tubeProgrammed reaction |
| spellingShingle | Matthew D. Fischer Emmanuel Mgboji Zhongchi Liu Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes Plant Methods Pyrite Cloning Restriction digestion Single tube Programmed reaction |
| title | Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes |
| title_full | Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes |
| title_fullStr | Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes |
| title_full_unstemmed | Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes |
| title_short | Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes |
| title_sort | pyrite cloning a single tube and programmed reaction cloning with restriction enzymes |
| topic | Pyrite Cloning Restriction digestion Single tube Programmed reaction |
| url | http://link.springer.com/article/10.1186/s13007-018-0359-7 |
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