Soybean promoter molecular cloning
- DESIGN: Each member was tasked with designing primers based on data we gathered through Integrative Genomics Viewer (IGV).
- BUILD: A construct was built through TA Cloning, allowing for digestion of the synthetic construct: the promoter region.
- TEST: Gel electrophoresis was run to verify amplification, cloning, and PCR was successful.
- LEARN: If the primers failed to amplify, we returned to the design phase and repeated until success was achieved.
Assembly of the vector construct
- DESIGN: The vector and each fragment was designed, and the approach of Gibson Assembly and Heat Shock Transformation was suggested for the fragments.
- BUILD: The vector was assembled and amplified within bacteria through PCR, Gibson Assembly, heat shock transformation.
- TEST: The assembly of the plasmid was confirmed through Colony PCR.
- LEARN: The result of Colony PCR determined whether it was necessary to repeat the cycle for this protocol. A successful result was achieved.
Identify genes low expression in seeds
Finding the promoter sequences from soybean genome:
What did we learn? What would we change?
The next cycle would be a bit different for us, as this would be utilized as a template, simplifying the protocol significantly. In a future iteration of the cycle, optimization of the protocol would include following our recorded successful protocol as closely as possible, given its success. We would also design multiple primers at one time in the instance one primer did not work, in order to be effectively prepared and save the time spent redesigning.