First week into the process of preparing nucleosomes

On Monday, Dr. Andresen and I began the process of preparing nucleosomes. We began with 50mL of chicken blood which if you’re wondering the reason behind why we use chicken blood, it’s because unlike our red blood cells, chicken red blood cells have nuclei that contain nucleosomes.

First off, Dr. Andresen and I began the process of preparing nuclei. We made 500mL of KTM buffer, which contains Tris HCL, KCl, MgCl2 and PMSF, and combined 50mL of it with the 50mL of chicken blood. Then we spinned that using a Centrifuge machine. The Centrifuge machine separates the heavier stuff (the pellet) from the lighter stuff (the supernatant) by increasing the gravitational force.Therefore, for the first couple of spins it helped us separate the red blood cells (the pellet) from the extra stuff that the blood contains such as plasma (the supernatant). Then, we resuspended the cells in KTM buffer and Triton X. The Triton X is like a “soap” which helps “break” the cells in order to release the nuclei inside of them. We had to do this because we want what's inside of nuclei, the nucleosomes.

Tuesday, we had a discussion about unproductive and productive stupidity in STEM. Right after the discussion, I actually made a mistake and added too much of one solution to a batch of KTM buffer that I needed to use in order to finalize the preparation of the nuclei. Dr. Andresen realized that I did this once he noticed how hard the pellet (which contained the nuclei) was since it wouldn't dissolve in the KTM buffer. Therefore, I had to start all over. I was frustrated at the fact that I had ruined a day of work but it actually helped me get over the fear that I had of messing up. It began my journey of understanding that research is a process of trial and error and that my mistakes will only help me learn and grow.

For the remainder of the week, Dr. Andresen and I went from having nuclei to having chromatin. When we had the nuclei, we measured how much DNA we had using the UV/VIS machine, which told us the concentration of DNA we had. We had about 125mg of DNA. Then, we resuspended the nuclei with CaCl2 + ML buffer, which contains Tris HCl, NaCl, MgCl2 and PMSF, and heated it at 37 degrees Celsius for 35 minutes. We heated this because throughout the process, the solution actually eats at the membrane of the nuclei which then breaks the nuclei and releases the DNA and everything else in the nuclei. Then, we continued by doing different steps in order to get the chromatin which consisted of making other solutions, spinning it, and letting it sit overnight with different solutions in order to get rid of the extra stuff such as ions and molecules surrounding the DNA.

As of today, Friday, we have chromatin, which consists of DNA linking nucleosomes together. Ultimately, what we’re trying to do is get the nucleosomes, which is an octamer wrapped around twice by DNA, so we'll have to cut the DNA linking the nucleosomes in order to get the nucleosomes alone. Also, today we measured how much DNA we have, which is approximately how much chromatin we have. We have 37mg of DNA which is about 30% of how much we had last time we had checked. We are hoping to at least have half of this amount by the end. However, during our group meeting today I found out that the next step is where things have gone wrong in previous years. Even so, I am hoping to have some nucleosomes next week.

Week #1: The Nano ITC

This week I've been spending a lot of time working with the Nano ITC, so I thought I'd take some time to explain a bit about how it works. After all, this is what I'll be using all summer!

Thar she blows! The Nano ITC in all its glory!

The Nano ITC, which stands for Nano Isothermal Titration Calorimeter, is a fun little box that can tell us all sorts of things about chemical reactions. In understanding what this instrument does, I find it useful to break things down word by word. First up is "Nano," which means it's pretty small. In fact, the ITC only uses about 350 microliters of sample per trial. That's about .0006 times the size of a Venti cappuccino at Starbucks! This lets us use a lot less sample to get the same information.

A view down the barrel of the ITC. At the bottom you can see the openings of the two chambers. The one in the center holds our sample and the one on the right holds our reference.

The second word in the name is "Isothermal," literally meaning "same temperature," which is exactly how the ITC works. Inside this box of wonder are two chambers, which are held at the same temperature throughout the experiment. As the temperature of our sample changes, it is checked against a reference solution placed in the other chamber, and the machine adjusts its temperature so the two match. The ITC measures the amount of power it takes to keep these two chambers at the same temperature, which gives us our results.

This is the syringe that the ITC uses to inject one of our solutions into the other.

The third word in the name is "Titration." This is a fancy chemistry word that basically means we're adding one chemical to another. The ITC uses a syringe to slowly inject small amounts of a solution into our sample. This causes a chemical reaction which we can collect data about. Also, we don't actually have to do anything after we start the machine, since the ITC does it for us! Finally, we have "Calorimeter." This just means that the ITC measures information about the thermodynamics of whatever chemical reaction is happening in the machine.
Putting all this information together, we see that the Nano ITC is a device that lets us measure thermodynamic data about a chemical reaction between two solutions. It does this by measuring the power it takes to keep our sample and a reference at the same temperature, and it doesn't even need that much solution to do it! And there you have it! That's a basic run-down of the Nano ITC. It's a really powerful tool that can give us a ton of useful information. Hope you liked the explanation, and I'll be back next week with some results from this week's testing!

First Week - Creating Nanoparticles

This week I started making gold nanoparticles (NPs) using two different methods, using citrate and CTAB surface coatings. The first is very easy to make, simply by heating up a gold solution until it boils and injecting trisodium citrate into it. This makes a red colored solution which contains the nanoparticles. The second method is much harder. The first step is preparing a "seed" solution that contains very small nanoparticles and then scaling these up in order to become bigger and easier to work with. Once the seeds have been made, they must sit for a few hours to grow and then the particles can be made by adding the seeds to a mixture of a gold solution, silver nitrate, and ascorbic acid. This is a very sensitive process, especially when adding the silver nitrate and ascorbic acid and can be messed up easily, which is what makes it difficult. However, if made correctly, the particles will be coated in CTAB and should be a red/pink color. The ones I made this week (two batches of 8 samples each) were relatively red/pink, but a few samples were purple/blue, which indicates aggregation (some of the particles have clumped together). This isn't what I want, as I want the particles to be as separate as possible in order to continue with my work wrapping DNA around each particle. If the particles are clumped, the DNA won't be long enough to wrap around and will cause problems with my experiments. I also made two batches of citrate-coated particles, which look a little redder than the CTAB ones.

Citrate-coated NPs. The middle is a nice red color, but the others are an ugly dark purple which indicates aggregation of particles.

When I ran my samples through the UV-Vis machine, which measures the absorption spectrum of the particles (how much of each color in the visible light spectrum is absorbed by the solution), most seemed decent, with a good solid peak of about 530 nanometers. This means that the particles are mostly absorbing green light / reflecting red light, which explains why the solution looks red. I had a few that absorbed way too much of other colors, so I'm not going to be using them to continue my research.

Absorption Spectrum from the first batch of CTAB-coated NPs.

I also ran my samples through a DLS machine which measures the size of the particles. Most of them were around 40 nanometers in diameter, which is a little on the large side, but it'll still work - I'll just have to correct for this when measuring how long the DNA should be. I had a few that were way too large, so I won't use these either.

The last analysis that I did is called zeta potential, and this basically measures the charge on the surface on the particles. Almost all of my particles performed well in this test and had the correct charge on their surface (about 30 millivolts). This is all well, however, the most important test is the absorption spectrum measurement, so all of the ones that did not do well in that test I will most likely be trashing. Next week, I'll start wrapping DNA around my particles and hope that all goes well - if not, I may need to create new particles.

Summer 2019 Begins with a Great New Crew

I would like to welcome Diana, Ben, and Nick, three wonderful new students to the lab. We have just begun our adventures together and already they are showing great promise. Keep checking back here for exciting developments as they tell us about their research!