I love my job

I managed to sequence through the important part of both plasmids. Sure enough, one of them showed a mutation at the 518 position just like it should have. The mutation was a serine to alanine. This means that at amino acid 518 we changed the peptide sequence from the wild type (serine - a phosphorylation site) to non-active mutant (alanine is not able to be phosphorylated). However, the other plasmid, which I thought was a wild type plasmid had a mutation at the 518 site that makes the protein always active. It is called a phospho-mimetic. That means that this mutation acts as though the protein was phosphorylated even though it is not. I need both of these constructs for the experiments that I have planned but I really wanted a wild type. Now I am going to have to go back and perform a site-directed mutagenesis reaction to change one of the plasmids back to wild type. The joys of molecular biology.

On Friday, Marlan and I did performed a couple of really challenging surgeries. One of the criticisms we recieved on our most recent grant proposal to the NIH was that the tumor placement in our mouse model (taking a brain tumor and placing it between the shoulder blades of a nude mouse) was not accurate. The reviewers wanted us to place the tumor in the mice in the same position as we find it in humans. It is hard enough to do skull base surgery in humans (from what I have observed) but to do it in mice would be quite the accomplishment. The first two mice we practiced on died. However, those failures enabled us to figure out the correct approach and to speed up the process. We managed to keep the last two mice alive throughout the surgery and through recovery. It was incredible. The surgery was performed under a microscope. By carefully exposing our site of entry, we were then able to drill out a small window in the skull, retract the cerebellum, and find the vestibular nerve which will be the area onto which we will place the tumor. This is going to be a cool experiment.