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.

Moving on

Sequencing is back. Although I haven't managed to sequence through the whole construct, what has come through is correct. What a relief. Only one more construct to do for this project. Let's hope it doesn't take me forever like the first two did.

OH YEAH!!

I'VE GOT A POSITIVE CLONE! I still need to sequence it but the banding pattern on the gel looks correct. I did a cartwheel in the lobby outside my lab yesterday.

Happy dance!!!!

I've got colonies! So I am doing a happy dance but with limited enthusiasm. Just because bacteria grew, doesn't necessarily mean that my plasmid is there. One of the reasons I am a little leery is that nothing grew on my positive control plates. That's not a good sign. I am going to go forward anyway and see what I've got.

No wonder I am going gray

I don't have to kick myself. The one thing that I thought would fix my Merlin problem, didn't. I figured that I was heat shocking the bacteria too long and that they were all dying. It made perfect sense. Yesterday, I decreased the time I heat shocked the bacteria but when I came in this morning, there was no growth on any of the plates. This afternoon, I am going to try a different approach. Instead of using heat to open up the bacterial wall, I am going to use electricity. If this doesn't work, I am pretty much up against a wall.

Dad, you said that you didn't understand what I have been talking about. Let me attempt to explain it. What I am doing is essentially editing. I am taking a sentance from one page, cutting it out of its context, and pasting it into a new paragraph with a new context. Instead of words, however, I am doing this with DNA. I begin with a process called PCR that "photocopies" the stretch of DNA I am in which I am interested (in this case, a gene called Merlin). This strand of DNA is then pasted (called a ligation reaction) into another strand of DNA (a circular contruct called a plasmid. Plasmids contain regulatory elements that allow my gene to be expressed efficiently) that has been cut open (this process is called a restriction digest) at sites that are complimentary to the ends of my DNA strand of interest. Now I use nature to make more copies of my finished product. I do this by introducing my plasmid into bacteria (usually a strain of E. coli) in a process called transformation. Finally, I plate my transformed bacteria onto agar plates and incubate them over night. The problem I have been having is that I am not getting any colonies on my plates, not even in my positive controls. Hopefully this explaination has been helpful.