Graduate School

I succeeded in implementing my mutation into my DNA. What this means is that I can now proceed onto the next step which includes making and purifying the protein. I am now on track to tag this protein with Nanogold and hopefully the next steps go well by the end of the year too. If not then we might have to change our plan and try another method. But I feel confident this could work.

I succeeding in purifying my DNA today! I followed the instructions more closely this time and got a really good concentration of 160 ng/ul. Now all that's left is to send it off to sequencing to make sure that my mutation took. If that works well then I can proceed with my project and get one step closer to wrapping it up and having publishable data. Fingers crossed.

Today was a much slower day. I was feeling really deflated from yesterday since I had failed at a task that is supposed to be the easy part. I know it's not that big of a deal but it just sucks getting it wrong from time to time. I started another overnight culture to try again tomorrow. That's pretty much all I did in lab today.

I attempted to isolate the DNA in my cells today and I got a really bad concentration. An ideal concentration is above 100 ng/ul but I acquired less than 2 ng/ul which makes it basically useless. I didn't know where I went wrong initially but I asked my PI to see what he thought I did wrong. There are several steps to the DNA purification process but the first step is to lyse the cells which means to break it up and get everything that's inside them out and floating in the solution. To lyse the cells we suspend them in 3 solutions, one at a time. After you add the second solution you are supposed to let it sit for 2-3 minutes. This is the solution that lyses the cells. It is imperative that this sits for the 2-3 minutes but I was adding solution 3 immediately afterwards. Solution 3 stops the lysing process and since I was adding it right away, most of the cells were not getting lysed. Oh well. Lesson learned, I'll try again tomorrow.

Good news today! I got growth on my plate. This means that I most likely incorporated my mutation successfully but I need to have it sequenced first to confirm. Tomorrow I will isolate the DNA and have it sent off for sequencing. If all goes well then I can continue with my project. If not then I’ll have to start over again. Fingers crossed.

Fingers crossed

The data shows that with the increase of phosphorylated tau, the global cognition of patients gets worse. The amount of phosphorylated tau increases over time and this leads to an increase in neuronal death. As neuron cells die, they spill the tau into the spinal fluid and it is this fluid that can tell us how far along in the disease a patient is.

The paper talks about how signs of Alzheimer’s disease can show up in brain scans up to 20 years before any symptoms kick in. I think it would be terrifying to know I’m going to get Alzheimer’s in a few years and there is nothing I can do about it. I’m getting my brain scanned every year from now on. Hopefully it’s not an expensive procedure.

It’s an interesting read and I’m glad I found this paper.

I continued the reading of the paper important for my research project. So far it seems really interesting. The goal of this paper is to label a protein at a desired Cysteine residue. It appears that many other groups before this one have been able to do so but when they did it, they relied heavily on good fortune to label the correct residue. However, this paper claims to be able to label a specific Cysteine using metal ions. I am interested to read more and understand how this works.

Today I prioritized finishing my homework before hanging out with friends. I feel proud of the fact that I was able to do that since I usually indulge in immediate gratification before delayed gratification. I also ordered some materials we need for the lab and for my specific project. No plasmids arrived this week which is disappointing but not a problem since it’s not urgent. Hopefully they arrive next week.

Today I made buffers to be able to purify my tau proteins. The buffers are essentially solutions in which the proteins can be suspended. The ionic concentration is important as it controls the levels of interactions between the proteins and other ions. This is a crucial effect in order for us to be able to purify proteins. I also ran a protein gel which separates the proteins in our solution by mass so we can see if our desired protein is in it. It looks like we do have a line where we need it to be and therefore we are on track to successfully purify K18 tau. Tomorrow we will continue the purification process and see if we can do it successfully.

I also continued on with my homework and made considerable progress. Still wish I didn’t have to do it but what are you going to do. The work continues.

Today I attended my Advanced Mechanics course. We are still doing mostly review material but it's quickly becoming more challenging than I initially thought it would be. I'm pretty glad about this though because I don't want to go to a class that is not mentally stimulating. I also started doing the homework with some classmates and this is also more difficult than I assumed it would be. I didn't miss the homework.

This development is useful because having a dense gold particle attached to the relatively "non-dense" protein allows us to "see" where the proteins are using cheaper imaging techniques like small angle x-ray scattering and dynamic light scattering. This is useful because with this technique we do not need to dry the solution and we can therefore observe the proteins in their "natural" state and observe its properties without much obstruction.

I took a break from research during the last month to study for my screening exam and I am very excited to get back to doing research again. I won’t find out my score until two weeks later so I’ll just keep myself busy until then so as to not worry about it so much.