Restriction digestion monitors facilitate plasmid construction and PCR cloning
Plasmid construction by “forced” or “directional” ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in ve...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2004-06-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04366ST03 |
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| author | Rishi D. Anand Odeniel Sertil Charles V. Lowry |
| author_facet | Rishi D. Anand Odeniel Sertil Charles V. Lowry |
| author_sort | Rishi D. Anand |
| collection | DOAJ |
| description | Plasmid construction by “forced” or “directional” ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a “monitor” plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination. |
| format | Article |
| id | doaj-art-39ad33787a274fda8e75a82c97e7c3b2 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-06-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-39ad33787a274fda8e75a82c97e7c3b22025-08-20T02:26:07ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-06-0136698298510.2144/04366ST03Restriction digestion monitors facilitate plasmid construction and PCR cloningRishi D. Anand0Odeniel Sertil1Charles V. Lowry21Albany Medical College, Albany, NY, USA1Albany Medical College, Albany, NY, USA1Albany Medical College, Albany, NY, USAPlasmid construction by “forced” or “directional” ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a “monitor” plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.https://www.future-science.com/doi/10.2144/04366ST03 |
| spellingShingle | Rishi D. Anand Odeniel Sertil Charles V. Lowry Restriction digestion monitors facilitate plasmid construction and PCR cloning BioTechniques |
| title | Restriction digestion monitors facilitate plasmid construction and PCR cloning |
| title_full | Restriction digestion monitors facilitate plasmid construction and PCR cloning |
| title_fullStr | Restriction digestion monitors facilitate plasmid construction and PCR cloning |
| title_full_unstemmed | Restriction digestion monitors facilitate plasmid construction and PCR cloning |
| title_short | Restriction digestion monitors facilitate plasmid construction and PCR cloning |
| title_sort | restriction digestion monitors facilitate plasmid construction and pcr cloning |
| url | https://www.future-science.com/doi/10.2144/04366ST03 |
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