Team:UCSF/Protocols

Cloning Overview

We cloned our protein into the plasmid by either using the restriction sites NotI-HF and BamHI-HF or by using homologous recombination. In the case of using restriction sites, we would first PCR the desired insert protein. We would then restriction digest the PCR product and the plasmid pCTCON2, separately of course. We would then phosphatase the pCTCON2. After we restriction digested both, we would PCR purify both the PCR product and the plasmid. We would then set up a ligation reaction. After we ligated the PCR product and plasmid, we would transform it into E. Coli Mach I cells. If successful, we would miniprep and transform into EBY100 Yeast cells.

In the case of using homologous recombination, we would make primers with the homologous region. We would PCR the desired protein and the plasmid with the correct primers. Afterwards, we would PCR purify and then transform it into EBY100 Yeast cells.