TY - JOUR ID - 6552 TI - Recombinase-free cloning (RFC) protocol for gene swapping JO - Molecular Biology Research Communications JA - MBRC LA - en SN - 2322-181X AU - Vo-Nguyen, Hai-Vy AU - Nguyen, Thanh-Tan AU - Mai, Quoc-Gia AU - Tran, Thien-Thien AU - Tran, Thuoc Linh AU - Tran-Van, Hieu AD - Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM AD - Department Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam AD - Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM Y1 - 2022 PY - 2022 VL - 11 IS - 1 SP - 21 EP - 27 KW - Cloning KW - Recombinant DNA KW - Molecular biology KW - E.coli DH5α DO - 10.22099/mbrc.2021.41923.1685 N2 - Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC). This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in E. coli host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories. UR - https://mbrc.shirazu.ac.ir/article_6552.html L1 - https://mbrc.shirazu.ac.ir/article_6552_d80e83e5be7c407e541979c39125a15c.pdf ER -