‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes
To clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. Some cloning vectors have been developed for the cloning of PCR products, including blunt-end and T-A cloning. However, different plasmids are required for the cloning of PCR products with blunt en...
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2024-12-01
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| author | Xingli Zhang Chong Teng Kaidi Lyu Shanhua Lyu Yinglun Fan |
| author_facet | Xingli Zhang Chong Teng Kaidi Lyu Shanhua Lyu Yinglun Fan |
| author_sort | Xingli Zhang |
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| description | To clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. Some cloning vectors have been developed for the cloning of PCR products, including blunt-end and T-A cloning. However, different plasmids are required for the cloning of PCR products with blunt ends and 3′ A overhang ends. Here, a novel cloning vector, pYFRed, which is based on the pUC19 backbone, has emerged and can be applied in both blunt-end and T-A cloning. PCR products can be cloned into the pYFRed by a one-step digestion–ligation reaction in a tube. The endonuclease recognition sequences of <i>Sma</i>I, <i>Eco</i>53kI, <i>Eco</i>RV, <i>Pme</i>I, and <i>Swa</i>I for blunt-end cloning and <i>Xcm</i>I for T-A cloning were designed and added between the <i>lac</i> promoter and the starting codon ATG of the <i>mScarlet-I</i> gene of pYFRed. The ligation efficiency was significantly higher because the restriction enzyme sites utilized were removed from the vector after being successfully constructed. The <i>mScarlet-I</i> gene was introduced into the pYFRed for the screening of the positive recombinants by the unaided eye without the need for additional reagents/equipment. pYFRed is easy to construct in an ordinary laboratory, which facilitates researchers to develop their cloning vector without purchasing commercial cloning vectors. |
| format | Article |
| id | doaj-art-1ff8d1f5b0144abba2353c2ed5c84baa |
| institution | Kabale University |
| issn | 1467-3037 1467-3045 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
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| series | Current Issues in Molecular Biology |
| spelling | doaj-art-1ff8d1f5b0144abba2353c2ed5c84baa2025-01-24T13:27:24ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452024-12-014711710.3390/cimb47010017‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided EyesXingli Zhang0Chong Teng1Kaidi Lyu2Shanhua Lyu3Yinglun Fan4College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, ChinaCollege of Agriculture and Biology, Liaocheng University, Liaocheng 252000, ChinaCollege of Agriculture and Biology, Liaocheng University, Liaocheng 252000, ChinaCollege of Agriculture and Biology, Liaocheng University, Liaocheng 252000, ChinaCollege of Agriculture and Biology, Liaocheng University, Liaocheng 252000, ChinaTo clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. Some cloning vectors have been developed for the cloning of PCR products, including blunt-end and T-A cloning. However, different plasmids are required for the cloning of PCR products with blunt ends and 3′ A overhang ends. Here, a novel cloning vector, pYFRed, which is based on the pUC19 backbone, has emerged and can be applied in both blunt-end and T-A cloning. PCR products can be cloned into the pYFRed by a one-step digestion–ligation reaction in a tube. The endonuclease recognition sequences of <i>Sma</i>I, <i>Eco</i>53kI, <i>Eco</i>RV, <i>Pme</i>I, and <i>Swa</i>I for blunt-end cloning and <i>Xcm</i>I for T-A cloning were designed and added between the <i>lac</i> promoter and the starting codon ATG of the <i>mScarlet-I</i> gene of pYFRed. The ligation efficiency was significantly higher because the restriction enzyme sites utilized were removed from the vector after being successfully constructed. The <i>mScarlet-I</i> gene was introduced into the pYFRed for the screening of the positive recombinants by the unaided eye without the need for additional reagents/equipment. pYFRed is easy to construct in an ordinary laboratory, which facilitates researchers to develop their cloning vector without purchasing commercial cloning vectors.https://www.mdpi.com/1467-3045/47/1/17cloning vectorPCR productsone-step cloningdigestion–ligation reaction<i>mScarlet-I</i> gene |
| spellingShingle | Xingli Zhang Chong Teng Kaidi Lyu Shanhua Lyu Yinglun Fan ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes Current Issues in Molecular Biology cloning vector PCR products one-step cloning digestion–ligation reaction <i>mScarlet-I</i> gene |
| title | ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes |
| title_full | ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes |
| title_fullStr | ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes |
| title_full_unstemmed | ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes |
| title_short | ‘Two in One’ Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion–Ligation and Screening of Positive Recombinants by Unaided Eyes |
| title_sort | two in one cloning vector applied for blunt end and t a cloning with one step digestion ligation and screening of positive recombinants by unaided eyes |
| topic | cloning vector PCR products one-step cloning digestion–ligation reaction <i>mScarlet-I</i> gene |
| url | https://www.mdpi.com/1467-3045/47/1/17 |
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