Golden Gate Cloning

Golden Gate Assembly

For my project, golden gate assembly is required to introduce homology arms which are specific to the genes of interest that I will be looking at in this project (SAS-4, SAS-6 and SPD-2). As we want to add HA tags to these genes to visualise their live translation and find to localise these genes, we need to introduce these HA tags to the genes first. To do this, I did golden gate assembly. Plasmids containing the 16 HA tags were already constructed and I had to now introduce these plasmids into my genes of interest.

To do this, homolgy arms (designed by my supervisor) were delivered and I first had to rehydrate these oligos with sterilised water to make 100 uM aliquots. As homologous arms were given as single strands, each arm had to be annealed together to make a double stranded homologous arm. This was done using the PCR machine (95C for 5 mins, then a gradual coling to 4C) - the first time ever I have used a PCR machine! These oligos were annealed together using a small volume of arms with an oligo duplex buffer.

Once double stranded homologous arms were achieved, the golden gate procedure can start. Here I mixed the homologous arms, the plasmid (with the HA GFP tags), ligase buffer, water and golden gate mix (containing the enzymes required to integrate the homologous arms into the plasmid). This mixture was heated for just over an hour, again in the PCR machine.

If all had gone to plan, my plasmids must now have the homologous arms on either side of the insert. The insert is now ready to be integrate into my genomes of interest!