Day 5

This morning I was pleased to find some colonies growing on one of the plates with the antibiotic on! This meant either, my experiment had worked and there were transformed yeast growing or some other contaminent was growing on it.

I was then shown how to make agarose plates containing more of the antibiotic. I was to pick some of the colonies and transfer some cells from each onto new plates, I also needed some more to replica plate the back up tranformation I began yesterday.

I spread cells from the 4 most visible colonies onto a new plate, I then decided to look at them under a microscope to make sure of what they were before moving on. It turned out that 3 out ofthe 4 colonies were yeast! The 4th however contained yeast and another smaller, stranger type of cell. This means my experiment may have worked in 3/4 cases and that I may have transformed yeast. I will have to discard the fourth however.

Once more colonies have grown over the weekend I will take some pictures. For now, the below diagram shows again what should have happened in the transformation. The old marker (G418) should have been replaced by a new marker (Hph). This was possible due to the upstream and downstream sequences of both being the same and so one should be able to replace the other.



There is still however a possibility that even if the transformation worked in the way that the new marker is now installed, that there is a chance the old marker has not been replaced. The old marker needs to be replaced for the further experiments taking place with this mini chromosome. To make sure it has been replaced another selection plate will be made up to see if the yeast will still grow on G418. If they do this means the yeast have both markers and the transformation did not work as we'd hoped.

Apart from this discovery I also got to watch some fluctuation tests, I think I understand them a bit better now. I will be doing them in future to observe rates of chromosome mutation like isochromosome formation.

There will be more on the new colonies and what they've turned out to be on monday!