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Digestions & Gels

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When we first started out our research this summer we were working on finding the best digestion for our chromatin. Digestion, simply put, is a way to get a fragment of a larger substance, in our circumstance, we are digesting chromatin to get nucleosomes. Our Senior Investigative Researcher (SIR) Macyn Rosay helped us to begin this process and slowly got us to do the process on our own. For the past week, we have been doing the digestion independently, looking for the best time for the 15-unit digestion. In the prior week, we had done a unit digestion that led us to 15 units the best for the digestion of our chromatin (so many gels). Unit digestion is how to find the baseline of Micrococcal Nuclease (MCN) to get the most amount of nucleosomes. From there we did a time digestion that ranged from 10 minutes to 55 minutes, and from this digestion, we concluded 30 minutes was the best for nucleosomes (see Fig. 1). Since 30 minutes was our blessed digestion we had (finally) moved to the next step which is mass digestion (only one more gel yay). The digestion process will be explained more in-depth later, for now, enjoy our fluorescent gel.

Figure 1: 15 unit Time Digestion of Chromatin

Once we (finally) made a good gel, we could move on. We spent the rest of the day digesting chromatin. We had a sample of 50 mL of chromatin that we separated into two separate test tubes with 25 mL in each just to make sure if we mess up (which we won’t), we are not using up all of the chromatin that we already have. If we do, then its back to the farm to get more chicken blood! 

We then added 4.5 mL of MCN to each of the two test tubes filled with chromatin and then centrifuged them. After that, we equally separated this new solution (chromatin + MCN) into 5 test tubes with 5 mL each and heated these samples for 30 minutes at 37 ℃. It was now time to make (another!) gel (and listen to Lana Del Rey of course).  Once 30 minutes had passed, we combined the 5 samples and added 1.25 mL of EDTA before putting it on ice for about 10 minutes (time for more Lana). 

Next, we prepared two samples for the gel, one with 5 μL of chromatin, 5 μL proteinase K, and 0.5 μL SDS. The other sample had 5 μL of digested chromatin rather than the undigested chromatin. We then placed these two samples into the isotemp for 50 minutes at 50 ℃. 

It was now time to move on to a higher-power centrifuge, which Professor Andresen taught us how to use. We added the digested chromatin to two centrifugal filter units and let those spin for about 10 minutes. In the meantime, we made a sample of 1.0 M NaCl which we later used to make 1.0 L of TEM buffer (Tris, EDTA, NaCl). 

We then finished the digestion and loaded the gel before letting it run for about 2.5 hours. While it ran we took a lunch break at the world-renowned Bullet Hole (it is THAT good). Once they were ready, we analyzed the gels and got the thumbs up from Professor Andresen (YAY).

Figure 2: Digestion of Original Chromatin & 30 min Digested Chromatin

So through some rough patches in the first few weeks, we made a breakthrough, but even when we were going insane we always had great music playing.

Signing Off
JIM 2 (Eden) and JIM 3 (Kyle)

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Week 1 (2024)

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The goal of the first week was to become familiar with GROMACS, the program used to computationally simulate processes of nucleic acids, and successfully simulate research with familiar results. After general tutorials and practices, I recreated part of the experiment done by Jejoong Yoo and Aleksei Aksimentiev in 2012 (“Competitive Binding of Cations to Duplex DNA Revealed through Molecular Dynamics Simulations”), where they added +1, +2, and -1 ions (Na, Mg, and Cl) to DNA in a box of water. The goal of these simulations is to first find the bulk concentration (the base number of ions surrounding the DNA) of each ion, and then the bound concentration. The bound concentration is found using radial integration of sections surrounding the DNA, and tells us how many ions cluster around the DNA.

Figure 1: Plot of the ion concentration when 25 Mg ions and 25 Na ions are added.

In Figure 1, we can see the bulk concentration where the concentration levels out around 25 A. The bound concentration is the area under the peaks. The graphs visualize the gathering of ions around the edge of the DNA. In this example, we can see that when there are equal amounts of Mg and Na, there is a significantly larger amount of magnesium clustered around the edge of the DNA. However, the next part of the experiment that I emulated was increasing the amount of sodium a significant more amount than the magnesium, which produced different results.

Figure 2: The graph from the original study that shows the Mg (red), Na (blue) and Cl (green) ions as the sodium concentration increases.
Figure 3: The graph made from our manual simulations of sodium, magnesium, and chlorine ions clustered around DNA.

Analyzing the graphs and the data points of Figures 2 and 3, we find that sodium and magnesium/chlorine have an inversely proportional relationship; as we increase the sodium concentration, the magnesium and chlorine charge decreases. This shows the “competition” aspect; when sodium increases, it pushes other ions away from the DNA. Comparing the figures, we can see that our simulation data matches the study’s results, which shows that we completed the simulations correctly and can trust our results. Now that we know how to simulate these processes accurately, we can move on to unfamiliar research.

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Repetitiveness of Research

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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.

Syringe from the ITC filled with clumped up DNA
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ITC Issues and Washing DNA

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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.

Macyn also posted our first lab Tik Tok! Go watch it at https://www.tiktok.com/t/ZTRoMh3t7/.

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Week 2 of Andresearch

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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.

Until I have to clock in again,

Macyn.

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Week 1 Update

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Hi everyone! I’m Aubrie, a rising sophomore physics major here at Gettysburg College. I’m excited to be working in Professor Andresen’s lab this summer for X-SIG and to work with my wonderful colleagues Macyn, Sofia, and Aston. My research is in collaboration with Macyn here at the beginning, and we will be researching the The Entropy and Enthalpy of DNA systems continuing from Tam’s work last summer. This past week we haven’t exactly started our individual projects yet, but we have started learning about the equipment available to us and how we might use it in conjunction with our research.

We started the week by learning proper pipette technique and practicing seeing our accuracy. It took some time, but I eventually was able to get the hang of it. I really struggled with the pacing of releasing the button, so I kept getting air bubbles in the tip which caused me to be under the expected amount. We also made salt stocks using NaCl and Cobalt Hexammine.

Tuesday involved us replicating an experiment that observed how DNA aggregates with Cobalt Hexammine. We learned how to make our DNA samples, put them through the centrifuge, and measured the concentration using the Nanodrop. We then added 20 microliters of Cobalt Hexammine and repeated those steps to see how DNA falls out of solution. We took a break in the middle to go for lunch at Food 101, but it was closed for renovations, so we ended up at Montezuma. I got the fajita burrito, and it was delicious.

We started Wednesday morning by making some DNA samples to use in the Circular Dichroism (CD) in the Science Center. We took an adventure to Prof. Buettner’s lab and got training on the CD, and later we ran our samples on the CD ourselves. I learned more about coding as well, and I feel a lot better about it already.

Thursday started out a bit rough. We were cleaning and ran out of water. A seemingly boring trip to get water ended up with a broken water jug and having to clean water off the floor. We were luckily able to borrow one from Prof. Thompson. Then we trained on the ICP with Prof. Andresen and that afternoon we trained on the ITC with Prof. Thompson. There is a lot to it, and we plan to replicate an experiment with it this week to see if we can get accurate results.

Friday was calm. I spent the morning reading a thesis from a former student and a paper that relates to it. I took some notes and googled a lot of terms. That afternoon we had personal meetings with Prof. Andresen to discuss how everything was going.

Monday has been pretty good. We started the morning with our group meeting, and I had some code issues, but it was a pretty easy fix. We discussed the best ways to read scientific papers as well. Macyn and I started a replication experiment with the ITC. Our first results were pretty rough, so we stopped the machine because we clearly made mistakes. Our second results were really good, and our third results were not consistent. We cleaned up after that one.

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Week 1 of 2023 Andresearch

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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.

Until another glass breaks,

                                             Macyn.

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Centrifuging and Washing DNA

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If you remember from my last blog post, we were about to embark on a new journey. After collecting a lot of data on zinc and what it does to DNA, we want to see if this change is reversible or not. Does the zinc bind so tightly that it cannot be washed away? Answering this question is what I’ve been up to this week. In order to do this, we took some samples of DNA in a 300mM zinc solution, and tried to get all the zinc out via centrifuge. We put the samples through a special filter that lets everything but DNA pass through, and then we add NaCl, MgCl2, and Tris buffer five times, running them through the centrifuge each time. After this was all done, I ran the samples through the CD spectroscopy machine, so we could see any changes in the DNA structure.

This is our latest graph, and we can see that DNA that has been washed with salts and a buffer looks very different from DNA in zinc solution that has not been washed. We can interpret this as the zinc unbinding from the DNA, which makes the peak at 275 nm revert back to how it was before zinc was added. However, we can still see some differences between the washed and unwashed DNA. The through at 245 nm is greater for the washed solutions, but is very similar for the DNA in plain water, and the DNA in zinc.

I am currently in the process of redoing this whole experiment. I hope that the new graphs will corroborate the data that I have already acquired, and help me analyze more accurately the differences between washed and unwashed DNA. Until then, I do think that we have some promising data, and I look forward to elaborate more on this experiment.

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Making Samples, Graphs, and Reading Data

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What have we been up to? For starters, the project about measuring Zn2+ binding to DNA has really taken off. We started by doing some samples of DNA in NaCl and MgCl2, which you might remember from our previous blog post. We did those several times to try to get some results that showed a change in the DNA structure. We ran all of our samples in the CD spectroscopy instrument, which measures how long it takes for DNA to unwrap. This time, we measured over the right span (320 nm to 200 nm), which means we can now read our data!

 What we wanted from these graphs was to observe a difference in the magnitude of the peak and trough of the graph, which was proportional to the sodium and magnesium concentration. In these graphs, it is possible to see that the concentration of the sodium and magnesium is directly proportional to the change in the DNA, however, we do have some outliers. Nonetheless, we decided we were ready to move on to the next step of the project, measuring the effects of zinc binding to DNA. We made two sets of similar Zinc and Magnesium samples, with varying concentrations, with the hopes of getting similar (but better) results to the ones above. We are doing both zinc and magnesium samples in order to compare the changes, to see if it’s the +2 charged ions that is causing the change, or the zinc itself. Then, we will do it all over again until we have a significant batch of good data.

We will also be looking to see if this change is reversible, or if the zinc binds so tightly to the DNA that it cannot be washed away. This will be achieved by exposing the DNA to zinc and later washing it with Tris, NaCl, and MgCl2.

Here are our latest results, which look very promising! We also learned a very handy tool to make our graphs look better and more accurate. By normalizing the line of each sample with the experimentally measured concentration of the sample, we are able to get rid of any variation read by the CD that comes with human error when making the samples.

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First Week Update!

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Hello everyone! Andresen Lab Crew Summer 2022 here! We are here to give you guys a rundown of our first  week of research. For starters, we are working with DNA (I know right!!). Aisha and Sofia will be collaborating on the same projects; Measuring the Kinetics of the Disassembly of Mononucleosomes and Measuring Zn2+ binding to DNA, while Tam will be independently working on The Entropy and Enthalpy of DNA systems.

The Golden Trio at Golden Hour

On our first day, we learned the basic skill of pipetting. We pipetted water in order to get a grip on the basics of pipetting before proceeding to pipette different solutions and liquids. Professor Andresen wanted us to use each pipette to measure the same amount three times, with the hopes of eventually getting a percent error below 5%.

On Tuesday we started the day by making DNA stock, which we would be using in all of our samples. We started out by measuring 0.08 grams of DNA. We put the DNA aside and pipetted (with our recently mastered pipetting skills) 7840 microliters of water into a 50mL tube. We then pipetted 80 microliters of TRIS solution and 20 microliters of EDTA solution into the tube, in order to make a TE buffer. We put the DNA in the tube and mixed it in the vortex mixer for a while, in order to break up most of the DNA. After that, to make sure the DNA was really broken up, we put it in the sonicator for several rounds. Now we had a 8mL sample of 10mg/mL of DNA, which we then diluted into a 1mg/mL sample so its concentration could be double checked in our spectrophotometer. After several rounds of checking the concentration, we got around 0.8 mg/mL, which is considerably less than what we theoretically had. However, the next morning we repeated the process and measured our concentration to be around 1mg/mL, so success!

On Wednesday, we started by mixing our DNA stock with NaCl and MgCl2 solutions. Our goal was to make 5mg/mL of DNA in different concentrations of NaCl and MgCl2. We used the formula M1V1= M2V2 (which looks the same as the momentum conservation formula) to find the amount of NaCl and MgCl2 we needed to get the right concentration for our solutions. After that, we had to dilute all of our samples by a factor of 5 because that was the concentration that our spectrophotometer can measure. Most of our results were within a reasonable range (the expected values of DNA’s concentration was 1000 ng/microLiter).

Today (Thursday), we spent the day in our collaborator, Dr, Buettner’s lab, using the CD machine to measure how long it’ll take for the DNA to unwrap, (the nucleic acid inside the DNA). After measuring all 11 of our samples, professor Andresen gave us a coding crash course, so we could start interpreting and graphing our results using Jupyter. However, we realized that we measured our DNA samples within a range that was too narrow, so we have to go back tomorrow and do it all over again. Nonetheless, we will not be taken aback by a small setback, we will come back stronger than ever tomorrow!