Welcome back to the Andresearch blog! This week hasn’t been very exciting, so sorry in advance if it seems repetitive, but when you’re doing research, all you do is repeat your work until it is good. The gist of the week was washing DNA samples to do more runs in the ITC.
One time of many of rinsing DNA through centrifuge filters
Wednesday, we learned new procedures regarding using the ITC. We now use a detergent called hellmanex to clean the cell because our samples are quick sticky and need a little more than water to get it out. We thought we had some really good data after changing the settings previously mentioned, and the extensive cleaning might have also helped. Our data looked pretty nice to us, but once checking the analysis software, it wasn’t what we were looking for. We discussed potential issues, and we decided to up our concentration of CoHex to 30mM.
You can see the viscosity of the DNA, and that is most likely why it sticks in the ITC
We plan to make DNA gels in order to find the length of our DNA, so we prepped for that by making 4 samples with varying dilutions. We had one at our normal concentration, and then a 10x, 100x, and 1000x dilutions. We made two things called ladders as well which you use to gauge the lengths after making the DNA gels.
When washing more DNA on Monday, we used a new type of filter, and our data was kind of weird. These new filters didn’t give us as much DNA than before, and we had to add more NaCl. We kept these in a separate tube to see if this poses an issue or not. This washing used up the rest of Batch One DNA, so on Tuesday we made Batch 2 after doing our ITC runs. Our ITC run seemed to give better results, but we were left with the DNA all clumped up in the cell as pictured below. We weren’t able to ask if this was what we were looking for or not, so we only did one run. For Batch 2, we followed normal DNA making procedure.
Happy Tuesday everyone! Friday was an uneventful day. We started off by cleaning the ITC from our overnight run and sending the data to ourselves so we could work on it in our lab. Cleaning the ITC involves grabbing a liter of Milli-Q Water and putting a needle attached to two hoses in the sample cell. The one hose connects to the liter of Milli-Q and the other to a flask that aspirates the water through the machine. While this was rinsing, we cleaned the injection syringe and the transfer syringe. Our data didn’t have a good baseline, and it was also missing solid peaks. After this, we washed more DNA samples to get them out of the TE buffer and put it in a Sodium Chloride buffer. We did this with 8 samples of 200 microliters of DNA in TE buffer.
This was our first DNA with CoHex injection run. After correcting the data by subtracting the baseline, we still were not getting correct results because the start doesn’t contain steady peaks.
Monday, we had the day off for Memorial Day. Everyone liked having a bit of a break, and I really enjoyed going home and seeing some family. Tuesday, we planned to run the ITC and have our weekly meeting. The time clock also came in, so we had fun messing around with that. It has a very satisfying stamp, so it was a necessary addition to our lab. We started warming the ITC up at 10, so we were able to use it by the time our meeting left out. The meeting involved discussing our progress and plans for the future. We then started our first sample in the ITC then and had 3 mg/mL DNA in 10 mM NaCl for our sample and did 20 injections of 2.5 microliters of our 10 mM Cobalt Hexammine in 10 mM NaCl. Our spin speed was 250 rpm, and the temperature was 25° C. We let this run while we went to our brown bag lunch meetings.
My lunch table had 3 psychology researchers and 2 biology researchers. The psychology researchers couldn’t divulge too much information about their research because they are hoping to use the other X-SIG researchers as participants in their studies, and we could mess up the data by knowing too much about the research beforehand. We are still in the beginning as well, so most of us are just doing the groundwork for our projects and getting trained properly.
After lunch, we returned to some not good-looking data from the ITC. We weren’t certain of what might have caused this, so we cleaned up and did a second run. While that was running, we deduced that it might be too high of a spin rate and that the sample was too small. Originally, we were using the standard spin rate of 250 rpm, but we are going to cut it back to 175 rpm which previous students used. The sample was too small because when it was drawn up into the syringe, air bubbles would enter because we tried to get the last drops from the sample. That is an easy fix though. We will just have to wash more DNA and put it all together.
This data looked promising at first, but it isn’t a good replica of the data collected before us. Since we are continuing a precious project, we need to get to where they were before we can start our research.
To start off this new week of research, we had a great team meeting to talk about our progress and, also, to discuss our goals for the week. For my partner, Aubrie, and me, our goal was to properly run the ITC. The Isothermal Titration Calorimetry is a process that involves keeping the equilibrium between a reference, which is usually water, and another sample that is being injected by another substance. The ITC then measures how the injections change the temperature of the reference and decide what it must take to keep the system (again, between the sample and reference) in equilibrium.
Very bad photo of the ITC. In the two small volumetric flasks are the EDTA and Magnesium solutions. In the current photo, the ITC is being cleaned, which involves a fun and only slightly loud aspiration process.
To train on the ITC, we tried to replicate the results of an experiment from a chemistry class taught by Professor Thompson. In this experiment, we injected EDTA, an acid, with magnesium. After a doing a total of ten runs of the experiment, we were able to get good data from four of those. The ITC is a particularly temperamental piece of equipment, which is why it can be so hard to get good data.
A graph of one of our test runs on the ITC. Each peak represents an injection of magnesium into EDTA.
Once we decided that we were trained enough to run the ITC, we began our actual project. For this, we would need to make some DNA solutions with a Tris-EDTA buffer. Fun fact: our DNA actually comes from salmon testes! Although, we are having a bit of trouble getting some new DNA (perhaps due to salmon shortages) after our last few orders were cancelled.
Since having a TE buffer in the ITC can cause problems, we needed to wash this DNA with the buffer that we would actually be using in the ITC; sodium chloride. To do this, we dispersed a bit of our original solution into smaller tubes and combined it with even parts of a NaCl solution we made. We spun this in our centrifuge, who is now named Spinch, for a total of three times before we were able to wash the DNA completely with the sodium chloride. Warning: there were more steps in this process, please do not try to replicate at home.
Another necessary ingredient for the experiment was a Cobalt Hexamine solution in sodium chloride. This turned out to be a very easy process and, before we knew it, we were ready to head over to the ITC and begin our very first run for our project!
For this project, we will be using DNA as the reference and the CoHex for the injections. We hope to replicate the data from last summer’s project as a starting point. We left the experiment running overnight and hope to see good results in the morning! For the last day of the week, we hope to run more of these experiments and try to problem-solve any issues that we may run into.
Professor Andresen’s lab is back up and running for the 2023 season and we have a great team joining us this summer! Sofia is returning to work on her project of Measuring Zn2+ Binding to DNA, while I have taken over Aisha’s project of Measuring the Kinetics of the Disassembly of Mononucleosomes and Aubrie has started work on Tam’s old project on The Entropy and Enthalpy of DNA systems. Also, Aston has joined the lab to work on Simulating Cobalt Hexammine3+ Binding to DNA. So far, things are looking great for this new team of researchers as they begin working in the lab.
First things first, the team learned expectations and safety measurements taken in labs. After, we all toured the different labs that we will be using, and learned about the important equipment that will be used in future experiments. Then, we all learned pipette techniques and how to properly handle basic lab equipment, such as tubes, flasks, and scales.
A partial view of the lab, displaying some pipettes and the scale used to make samples.
We then made salt solutions using all the techniques we were just taught. Surprisingly, we only broke one volumetric flask! Fortunately, this was the perfect chance for us all to practice safety while cleaning up glass and how to properly dispose of it. What a great learning experience!
On Tuesday, we replicated a DNA aggregation experiment. To start, we made a solution of DNA with a buffer, water, and a pinch of salt—a great recipe if you’d like to follow along at home. In our experiment, we gradually added more and more Cobalt Hexammine to our DNA solution and recorded the aggregation of the DNA.
The Cobalt Hexammine sample we made for use in other experiments.
To record such data, we learned about and used the NanoDrop. The NanoDrop is a special measuring tool that takes in only a little bit of the sample and can tell how much the DNA has aggregated.
Data taken from the NanoDrop to make a graph of the DNA Aggregation for the first sample of DNA with 20 microliters of added Cobalt Hexammine.
We made two samples to record our data and we were able to replicate the results of the experiment.
A graph of the two samples’ DNA concentration measured against added Cobalt Hexammine.
We can see from the graph that the concentration of DNA decreases as the amount of Cobalt Hexammine (CoHex) is added to the samples. These are the results that we expected to see in our experiment, as they match the results begotten from the original experiment that we were replicating. Also, on Tuesday, we all had lunch together at Montezuma and it was very fun. I would highly recommend it.
Wednesday, we made three samples of DNA solution with increasing amounts of Zinc. Our goal was to use CD (Circular Dichroism) Spectroscopy to see the changes in the structure of DNA.
Sideways view of the CD Spectrometer, otherwise known in the Science Center as DJ Polarizer.
The CD does this by shining polarized light through certain molecules at different wavelengths. We were trained on the CD, and then we learned how to use Jupyter to plot our data and better analyze it.
A graph of the shape of DNA measured by the change of ellipticity over wavelength.
Our graph shows the shape of DNA with varying levels of Zinc added. A baseline` of water can also be seen. We can clearly see that DNA’s shape is affected as more Zinc is added, though the general shape of the graph remains the same.
On Thursday, we got trained on both the ICP and the ITC. Before all that, though, I washed a few dirty dishes in the lab, because a clean lab is a good lab! However, I noticed that our water was running low, so Aubrie and I went to go get some more. Unfortunately, we soon realized that our tank was cracked. After making a few small puddles in the lab, we borrowed another tank and replaced the old one. Then, we were able to move on to training. The ICP, which we got trained on first, is the Inductively Coupled Plasma Optical Emission Spectrometry.
View of the ICP.
It takes a sample and sprays it into plasma, which then breaks chemical bonds and excites the electrons in the element. When the electrons move states, they emit light. The machine takes in this light’s wavelength and can tell, since this wavelength is element-specific, which element is in the sample. Next, we got trained on the ITC, or the Isothermal Titration Calorimetry, which measures the binding constant, the enthalpy, and the stoichiometry of samples. We can also use these measurements to calculate free energy and entropy. We also held a milk and cookies social for all the researching students on campus, which was a ton of fun!
We all look forward to the rest of our week and the rest of our experience this summer! We hope to make great progress in all our projects and, of course, have a decent amount of fun.
