All-in-One Molecular Cloning as a New Gene Manipulation Method

DNA cloning plays a crucial role in biotechnology laboratories and biotechnology-related fields, which facilitates the primary yet crucial step for further studies in molecular biology. Many laboratories worldwide still use restriction enzyme-based cloning methods to construct expression vectors owing to financial constraints, time-consuming, and the unavailability of appropriate vectors. In the present study, we introduced a novel method inspired by the restriction enzyme-based cloning method with some modifications named All-In-One (AIO) cloning.

The PCR product and vector for cloning were digested in one 0.2 ml tube with a total volume of 20 µl. Completely digested products were checked and inactivated by heat treatment. Digested genes and vectors were directly used for the ligation step in this 0.2 ml tube, without any purification step required. Finally, ligation products were transformed into competent E. coli DH5α by the heat shock method.

More than eight different clones were generated by using AIO cloning, which all the necessary reactions were performed in one single 0.2 ml tube. This method was efficient in cloning a wide range of DNA fragments, from 200 to 1300 bps.

Collectively, AIO provided an alternative yet sufficient cloning protocol, reducing the loss of DNA components, and saving materials, labor and time, especially where research materials were not abundantly available.