Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly
Abstract Background The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2021-03-01
|
| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12896-021-00679-6 |
| _version_ | 1818610649369935872 |
|---|---|
| author | Fan Chen Yi-ya Li Yan-li Yu Jie Dai Jin-ling Huang Jie Lin |
| author_facet | Fan Chen Yi-ya Li Yan-li Yu Jie Dai Jin-ling Huang Jie Lin |
| author_sort | Fan Chen |
| collection | DOAJ |
| description | Abstract Background The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. Results We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. Conclusions Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies. |
| first_indexed | 2024-12-16T15:17:47Z |
| format | Article |
| id | doaj.art-117bc09b75f84b91b0ab500b9bc7422a |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-16T15:17:47Z |
| publishDate | 2021-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-117bc09b75f84b91b0ab500b9bc7422a2022-12-21T22:26:44ZengBMCBMC Biotechnology1472-67502021-03-0121111310.1186/s12896-021-00679-6Simplified plasmid cloning with a universal MCS design and bacterial in vivo assemblyFan Chen0Yi-ya Li1Yan-li Yu2Jie Dai3Jin-ling Huang4Jie Lin5School of Biological Science and Biotechnology, Minnan Normal UniversitySchool of Biological Science and Biotechnology, Minnan Normal UniversitySchool of Biological Science and Biotechnology, Minnan Normal UniversitySchool of Biological Science and Biotechnology, Minnan Normal UniversitySchool of Biological Science and Biotechnology, Minnan Normal UniversitySchool of Biological Science and Biotechnology, Minnan Normal UniversityAbstract Background The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. Results We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. Conclusions Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.https://doi.org/10.1186/s12896-021-00679-6Plasmid cloningUniversal MCSBacterial in vivo assemblyDNA assemblyHomologous sequence |
| spellingShingle | Fan Chen Yi-ya Li Yan-li Yu Jie Dai Jin-ling Huang Jie Lin Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly BMC Biotechnology Plasmid cloning Universal MCS Bacterial in vivo assembly DNA assembly Homologous sequence |
| title | Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly |
| title_full | Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly |
| title_fullStr | Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly |
| title_full_unstemmed | Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly |
| title_short | Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly |
| title_sort | simplified plasmid cloning with a universal mcs design and bacterial in vivo assembly |
| topic | Plasmid cloning Universal MCS Bacterial in vivo assembly DNA assembly Homologous sequence |
| url | https://doi.org/10.1186/s12896-021-00679-6 |
| work_keys_str_mv | AT fanchen simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly AT yiyali simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly AT yanliyu simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly AT jiedai simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly AT jinlinghuang simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly AT jielin simplifiedplasmidcloningwithauniversalmcsdesignandbacterialinvivoassembly |