Cloning short DNA into plasmids by one‐step PCR
Background Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐s...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020-11-01
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| Series: | Thoracic Cancer |
| Subjects: | |
| Online Access: | https://doi.org/10.1111/1759-7714.13660 |
| _version_ | 1818521511321927680 |
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| author | Cheng‐Cheng Tao Ying Yang Fang Li Ling Qiao Yue Wu Xiao‐Dong Sun Yuan‐Yuan Zhang Chang‐Long Li |
| author_facet | Cheng‐Cheng Tao Ying Yang Fang Li Ling Qiao Yue Wu Xiao‐Dong Sun Yuan‐Yuan Zhang Chang‐Long Li |
| author_sort | Cheng‐Cheng Tao |
| collection | DOAJ |
| description | Background Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo. Methods The pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy. Results The pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells. Conclusions Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction. Key points Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids. |
| first_indexed | 2024-12-11T01:52:02Z |
| format | Article |
| id | doaj.art-ff4d55f3524640adaad89516f231a6d8 |
| institution | Directory Open Access Journal |
| issn | 1759-7706 1759-7714 |
| language | English |
| last_indexed | 2024-12-11T01:52:02Z |
| publishDate | 2020-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | Thoracic Cancer |
| spelling | doaj.art-ff4d55f3524640adaad89516f231a6d82022-12-22T01:24:44ZengWileyThoracic Cancer1759-77061759-77142020-11-0111113409341510.1111/1759-7714.13660Cloning short DNA into plasmids by one‐step PCRCheng‐Cheng Tao0Ying Yang1Fang Li2Ling Qiao3Yue Wu4Xiao‐Dong Sun5Yuan‐Yuan Zhang6Chang‐Long Li7West China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaWest China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaDepartment of Medical Oncology, Sichuan Cancer Hospital and Institute, School of Medicine University of Electronic Science and Technology of China Chengdu ChinaSchool of Basic Medicine Chengdu University of Traditional Chinese Medicine Chengdu ChinaWest China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaWest China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaWest China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaWest China School of Basic Medical Sciences & Forensic Medicine Sichuan University Chengdu ChinaBackground Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo. Methods The pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy. Results The pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells. Conclusions Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction. Key points Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.https://doi.org/10.1111/1759-7714.13660Homologous recombinationone‐step PCRplasmid constructionrestriction enzyme‐based cloning method |
| spellingShingle | Cheng‐Cheng Tao Ying Yang Fang Li Ling Qiao Yue Wu Xiao‐Dong Sun Yuan‐Yuan Zhang Chang‐Long Li Cloning short DNA into plasmids by one‐step PCR Thoracic Cancer Homologous recombination one‐step PCR plasmid construction restriction enzyme‐based cloning method |
| title | Cloning short DNA into plasmids by one‐step PCR |
| title_full | Cloning short DNA into plasmids by one‐step PCR |
| title_fullStr | Cloning short DNA into plasmids by one‐step PCR |
| title_full_unstemmed | Cloning short DNA into plasmids by one‐step PCR |
| title_short | Cloning short DNA into plasmids by one‐step PCR |
| title_sort | cloning short dna into plasmids by one step pcr |
| topic | Homologous recombination one‐step PCR plasmid construction restriction enzyme‐based cloning method |
| url | https://doi.org/10.1111/1759-7714.13660 |
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