Each good project needs a good organisation and strategy. For a better understanding about what and why we did in our laboratory, we created this flowchart with a detailed overview.
First of all we planned which genes and homologue regions we wanted to use and we designed their respective primers using Geneious . For this process we used always this common pattern
The most important characteristic of the designed primers for the production of the DNA Fragments was the use of overhangs. With this primers with overhangs we generate the desired fragments with homologous parts.
After the production of all desired fragments we assembled them using the Assembly Kit from New England Biolabs. Afterwards we verificated the assembly product with PCRs, electrophoresis and sequencing.
With the already good assembled fragments we transformed the yeast strains using the Frozen-EZ Yeast Transformation II Kit from Zymo Research.
To review the transformed yeast we isolated their genome with the Quick and Dirty protocol and after a positive verification-PCR with the respective primers, the sequencing of the introduced gene was given in order to Eurofins.