Category Archives: Week 4

A Day in the Life

For me, each day of the week in the Caron lab is dedicated to a different procedure with the overall goal of generating data for my project. Thanks to my dedicated mentor who goes to lab on Sunday, I am able to run three rounds of BRET in a week.

On Mondays, I transfect the cells that my mentor split the day before. I also split cells for transfections on Tuesdays. On Tuesdays, I split the transfected cells to a 96 well plate for BRET and transfect the cells split on Monday. On Wednesday, I am able to finally use the BRET machine and see if my transfections and plating reveal any interesting results. Like is expected for good science, I try to achieve many rounds of similar results with BRET assay to assure that the data is not due to error on my part. Thus, I run BRET on Thursdays and Fridays as well.

While the BRET assay is my main objective in lab, it is not the only thing I do. Each time I transfect, I have to use some DNA from the stocks we keep in the fridge. After many rounds of BRET, the DNA starts to run low. Therefore, I occasionally engage in cloning projects in order to make more DNA. Sometimes, I am lucky that other people in the lab have glycerol stocks of the DNA I need. If so, I am able to utilize a Qiagen MidiPrep in order to extract more DNA. However, if no glycerol stocks are available, I have to transform the DNA in bacteria and let the bacteria grow up copies for me.

In addition to all of these small mini cloning projects, I did partake in a fairly lengthy cloning project that lasted an entire week. I attached a fluorescent probe to the D3 receptor.

So, this is how I have been keeping busy in lab. Sometimes, if I finish everything I have to in a day (or if there ends up being too few cells to split), my mentor will keep me busy with random projects that usually involve new techniques. One thing is for sure, there is never a boring day in the Caron lab.

It’s about time…management

My day usually starts with me trying to drag myself out of bed…which takes a while.
Then I go to the program meeting or have breakfast. Before work or at least sometimes during, I usually have some sort of coffee, which is very unlike me. But ever since I came here, I’ve hopped on the coffee train and can’t seem to get off.

My time in the lab is usually spent planning tests, coming up with ways to change variables, and checking for transformants. Recently I’ve been repeating experiments to see if we get faster/better results. For instance, it took a while for us to find definite transformants in the last set of plates we shot with DNA, so I redid the experiment with twice the amount of DNA and different goldbeads.

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I’ve also developed improved techniques for transferring cells to drug plates. I’ve wrapped up the last week by streaking more drug plates in different sections to test for growth. If they grow, it confirms that we actually do have transformants. I’m very excited to check today if we have any!

Link 

After work, I usually rush to buy food before everywhere closes (I leave work around 7 or 8, or sometimes 9pm). And I spend the rest of the day relaxing in prep for tomorrow so that my mind is clear. Sometime I get to leave work early and I go to the gym 🙂 But I’ve recently been going to the gym on the weekends, after I’ve run a few errands.

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My days are usually longer than I anticipate, so I think what I would love to accomplish before I leave is better time management.

 

A typical lab day

My day in the Levin lab usually varies. Some days I come into the lab and I have a few rats to harvest. Harvesting includes extracting the brain and spleen and liver from each rat (after they have died of course). Sometimes I am also in charge of putting the rats through the series of varies tests that we give them as mentioned in the protocol. These tests include tings like attention tasks, figure 8  mazes, and item recognition test.

The other half of my time in the lab is spent doing less glamorous things like feeding the rats, changing cages, washing cages, marking their tails and weighting them, and also getting bit every once in a while. But everything that one does in the lab cannot be completely glamorous. I am very glad that I get to do interesting things when the opportunity comes and the days usually go by pretty fast. My lab also uses Zebrafish but I never work with them. I usually see them only in passing.

Sometimes, we get fire drills because the cage wash causes the fire alarm to go off every once in a while and everyone has to stop what they’re doing to go outside and wait for the fire department to come. They usually look kind of annoyed. OOPS.

Well, that is usually some of the things that my days in the lab entail. I’ll be learning how to do surgery on the rats soon which might be fun.

Thanks for reading!

Syringe. Clean. Aspirate. Inject.

In four actions, that is essentially a day at work for me. Let me clarify, that’s just the series of actions you go through when you’re running samples through High Performance Liquid Chromatography (HPLC). I’m essentially a lab assistant and in a day of work I help around lab, clean, and run samples through HPLC. I’ve also helped in lab with an unexpectedly biological aspect: the sacrifice and dissection of rats and rat brains, respectively. I want to talk about the two main contributors to my day in lab but also discuss how I’m still growing by working in lab.

HPLC is an analytical chemistry technique used to separate analytes of interest in a solution and analyze them. Paired with an electrochemical detection (ED) system and computer software it can visualize the presence of your analytes as peaks on a chromatogram and estimate concentrations. This technique is simply column chromatography performed under pressure. Simple column chromatography is a column with some sort of “stationary phase” particles through which a liquid mobile phase is passed through at the same time a sample is in the column. A combination of the interactions of the analytes with the stationary and mobile phases will determine the order in which the analytes are eluted, or released. HPLC is this in essence except in order to achieve effective detection of little analyte, it is run under pressure. The applied pressure pumps the mobile phase through the column of stationary phase and a sample is ejected in. This is the most tedious part of the day for me. The apparatus just runs for hours when the pump is turned on and it is just a matter of constantly taking a syringe, cleaning it, aspirating sample, and injecting it. Though tedious, it is a necessary process. Further, the interesting components of HPLC is the ED system.

The ED system works through an electrochemical cell with working, auxiliary, and reference electrodes. As analytes pass through the column they diffuse through into the cell and undergo an electron transfer reaction, an oxidation or reduction reaction, electrons are transferred and detected by the working electrode, and the signal appears as a peak on a chromatogram through a computer program. Almost exclusively because of the electron absorbing properties of the working electrode, an oxidative reaction is the preferred electrochemical reaction to occur after diffusion of analyte into the electrochemical cell. HPLC conditions such as the pH of the mobile phase and the temperature used to run the apparatus are tailored to favor oxidative reactions. The peaks are further processed using computer programming to determine concentration of analytes by comparison with a pre-made standard curve. This final step wraps up of the process of HPLC paired with an ED system. What is left to be discussed is the animal handling part of my work before moving onto what I have gained from these experiences.

A total of three times, I have helped in the process of harvesting blood and brain samples from male and female rats. We begin by weighing the rats and performing one of three treatments on them: 1) gavaging them with the RTD (Rapid Tryptophan Depletion) amino acid (AA) solution, 2) the BAL (balanced AA solution with all essential AAs including tryptophan), or 3) treating them with neither solution. We euthanize the rats in a bell jar to the point that they will not suffer during the sacrifice. The brain is extracted from the cranium and dissected for predetermined brain parts. The blood is also centrifuged for the extraction of plasma and the brain is stored before being sonicated later and centrifuged for analysis. Both the plasma and brain samples are run by me and my lab supervisor, Zack Reavis, through HPLC for sample analysis.

These two processes are essentially tedious yet necessary for the continuation of progress in lab. Sometimes it does become quite drab and it really starts to feel like I’m working a glorified 9-5 job. However, I quickly correct myself. I cannot take for granted just how much I have learned in lab, how many problems I’ve encountered that I’ve had to actually think about to solve. My PI was so particular about designing a project that would somehow allow me to encounter a problem essentially chemical in nature for me to solve and actually do some chemistry! I helped adjust a protocol such that it was tailored for our needs during sample analysis: adjusting the pH of the mobile phase such that the eluted analytes came at a rate that would produce a clean chromatogram with nicely separated peaks. I need to appreciate that even though I am completing a project here that will allow me to experiment to answer a question I’m mostly here this summer to learn what it is like to work in a lab environment, learn some lab techniques, and just get a feel for the life of lab work. This is exactly what I have gotten to do and I’m getting quite a good feeling about what it’s like working in the lab. I’m beginning to think this is something I might want to do for the rest of my life.

Is Every Day the Same?

After completing my fourth week of the BSURF program, I have a better understanding of my role and what to expect in the lab. Most of my time in the lab revolves around a computer screen, while a small portion of my time is spent doing hands-on things. But my lab mentor tells me that thorough background research, careful planning of experiments, and careful data analysis take most of a bench scientist’s time, and not the actual bench work running the experiments. This ascertains that the best science possible is being done, to obtain data in a purposeful and hypothesis-driven manner.

With my project, there is a great emphasis in studying inflammatory breast cancer in an in vitro model. My time spent doing hands-on protocols revolve shadowing the graduate student, Risa, producing media for cells, cell culturing, splitting cells, and imaging the progression of tumor emboli via a microscope.

As mentioned earlier, most of my time is spent in front of the computer screen. I am in front of a screen because that is where the data is kept. Using computer software is how I can calculate the size of the tumor emboli growth quantitatively and use that information to produce graphs. However, graphs are tricky, because there are multiple of ways of interpreting the data depending on how you choose to represent and graph the data, and what you are comparing. Furthermore, what makes the process more complicated is finding the right way to convey the information to others, in order to produce a visual result that speaks to the postulates made during hypothesis formation. The foundation of science is built around discovery and sharing those discoveries with the public, and therefore the information scientists choose to share should be able to be understood by others, even sometimes intentionally by the general public. These past few days I have spent a lot of time ensuring that the way I chose to present my data is visually easy to understand and is able to get the point across that I am trying to make. There are some statistical analyses that we have done as well with the graphs to further speak to the significance of the results and trends we have identified from the data.

I am enjoying my time in the lab; I feel that I now understand more of the molecular and biological reasoning behind my project than I did in the beginning. Most of my days may be in front of a screen, but each day there are advancements in my project and I find out more about the data I have or find better ways to present my data to other people. What I have learned so far with this experience is that there is so much data on science projects out there, but it takes time to interpret it correctly. Hopefully, by the end of this program, I will be able to share my data in a way that is meaningful and easily related to my audience.

A Day in the Life

My day usually starts with cells. I am culturing dendritic cells, and they need to be passaged on Monday, Wednesday, and Friday. So when I get into lab, I meet with the Lucas, the graduate student I am working with, and we talk about the plan for the day, and then I usually passage my cells shortly after we meet. On Tuesday and Thursday I just take a look at my cells under the microscope to make sure they are still growing healthily. Then I launch into the other tasks of the day.

For the first couple weeks, a lot of what I was doing was learning procedures and finding my way around the lab. I would watch Lucas do a variety of tests and experiments, trying to absorb everything that was going on. I learned how to passage my cells, do ELISAs, stain histology slides, and more. I am now doing more and more on my own now, becoming more confident around the lab. What I do can vary a lot from day to day depending on where Lucas and I are with our experiments, but it all centers around the same main experiments. I split my cells either just to passage them or to prepare them for an experiment. If an experiment is coming up, I will do other prep work for the cell experiment, and if it’s the day of an experiment I will perform the experiment. I may cut lymph node tissues, stain the tissues, or look at the stained tissues under the microscope. And, if it is the day of a blood draw, I might do an ELISA.

One thing that is a constant every day, though, is that whenever I am not doing anything at the bench, I am reading. I have done a ton of reading in the last month and I have learned so much about immunology that I would have probably never learned any other way. I started off by reading the better part of an immunology textbook, and then moved on to reading articles about biomaterial vaccines and engineering the immune system. I have a whole folder of papers on my computer of papers to read, and am making my way through them one by one in my free time. So far, I am enjoying my time in the lab a lot and I am learning so much every single day.

Waking Up with a Purpose

The impending doom of an alarm clock waking me up from my nightly hibernation is less terrible when I know I have lab to look forward to (needless to say I’m not a morning person). Usually three snoozes later my day will start around 8:15. I’ll gather my things and make the arduous trek up the Edens stairs to my happy place: Joe Van Gough. The heroes of JVG will provide me with my elixir of life and I’ll finally start to come out of my hibernation.

Walking into lab (full of coffee, of course) is always incredibly exciting. I’ll never fully know what the day entails, but I know I’ll always get to learn something. For the past ten days we’ve been doing the social defeat experiment at 11 AM. I’ll spend my morning getting to hold cute little mice. I’ve been known to name them, cuddle them, and maybe drop a few ‘I love you’s if I’m feeling extra perky. I get to do science while spending time with animals and some great lab members. After the experiment is over, we usually break for lunch. I’ll hike my way to West Union and battle my way through 12 year olds to get food.

Back in lab, I’ll see what other lab members are doing and see if they need help. My official work for the day is over, but my lab usually needs help doing histology. We implant electrodes into the brains of the mice before our social defeat experiments to get data about activity in certain brain regions involved with depressive-like symptoms. To validate our data we must look at the brains after the experiment to make sure the electrodes we implanted were in the right brain region. That way we can support that our results show data from the region that we intended to be recording. I love this portion of the day because I get to see the project I’m currently working on come full circle. There’s always more histology to work on so I get to learn something new every day.

After my histology is done I make the walk back to Edens and spend my night finishing a paper for lab. I’m extremely grateful that I get to work every week with such talented lab members and have the privilege of learning from them. Not many undergrads get to learn so much so early and truly experience what life in a lab is like.

Learning Lab Life Lingo

That’s a lot of L’s.

Not to worry, the L’s are an unfortunate pattern, unrepresentative of this blog post. If you can think of synonyms beginning with, let’s say the letter, W, feel free to comment below.

At this point, I’ve grown much more accustomed to lab life and the basic, daily lingo. Four weeks ago, if someone were to ask me, for example, to split a certain cell line 1:10 onto 3 plates, passage cells once they attain 90%-100% confluency, or, as I perform a western, to dilute a primary antibody 1:5,000 because it produces relatively brighter bands, that person would receive a blank, somewhat bewildered stare. These fundamental concepts of lab life have already become so ingrained into my brain that my lab mentors trust me to do various activities independently such as taking care of my cells and performing western blots. There have even been days where they let me loose the whole day to conduct experiments they had previously taught me. Of course, as the learning process works, I would still ask little, clarifying questions, but I felt like a real scientific researcher confidently walking around with protein samples in hand, instead of following around another lab member.

I’ve never done the same exact activities in lab that I’ve done in a previous day. That is a beauty of research. I don’t go to work expecting to perform the same routine from 9am to 5pm every day. The timing is dependent on the experiments planned for the day. Future experiments are also determined by the results of experiments I do now or have done in the past, and that is exciting. Unpredictable in a sense, which makes lab life exciting. Yes, there are some activities involving taking care of my cell lines that I must attend to every day. However, even the timing for cell passaging and changing media varies day to day, depending on the times that other lab members sign up to occupy the hood.

Something special I’ve noticed about the culture of my lab: we help each other out. I experience this nurturing culture not only in the weekly 2-hour lab meetings, but also in the day-to-day scene. If a lab member working primarily with breast cancer cells needs a lung cancer cell line, another lab member who works primarily with lung cancer cells freely provides a plate of cells from the desired cell line. If a lab member needs to split transduced cells over the weekend, another lab member who has to come in during the weekend anyways graciously offers to split the transduced cells so that the other lab member can take the weekend off. If a lab member needs to leave lab for several days for personal reasons, another lab member is happy to take care of his or her mice in spite of all of the deadlines that need to be met or experiments that need to be run. Cells, reagents, antibodies, and information flow freely throughout the lab. Sometimes while I’m just sitting at my desk, the postdoc in the lab casually says, “Hey Kristie, want a pathology crash course?” or “Hey Kristie, want to watch me harvest mouse lungs?” One minute later, he is showing me on his computer cross sections of the left lung of a mouse that had pneumonia, explaining the versatility of the organ of the lungs, or teaching me the scissors dissection technique of separating tissue by opening the scissors, not cutting down.

The thing about lab life is, I could see myself doing this every day. Working in a lab 10-hours a week during the school year really is not the same as the immersive experience of a summer of research where I can experience every step of the research process. The research I conduct in this lab is still, surprise, fascinating! It is incredibly intellectually stimulating. The excitement from obtaining results and being the first to analyze them is an unparalleled sort of excitement because the reality is, researchers often deal with new information that no one other than themselves has ever produced before. As a researcher, I am not bound by strict time constraints or the same routine every day, but rather I am free to explore the unknown and continually learn and share new, up-to-date knowledge. I could get used to the exciting life and open community of scientific researchers. I really could.

The Excitement of a Day in the Lab

I wake up by 8:00 every morning just in time to get my morning coffee, making sure to start my day off with a kick. Depending on the day I will either head to French Family Sciences for a B-SURF meeting or I will head straight to my lab in CIEMAS.

I will then either begin my day in the lab with a paper relating to my lab’s research or I will get straight to business, working on research.

My day-to-day research varies immensely depending on what my mentor, Nikki, is working on for the day. Many of her studies involve working with mice to study immune responses elicited by various copolymers, so we spend a lot of time prepping vaccines for the mice and running ELISAs to analyze the elicited immune responses. I spent much of the first couple of weeks shadowing Nikki as she taught me some of the various techniques her research entails and as each week passes by I get opportunities to do more and more techniques on my own.

Once it gets to be around lunchtime I take a coffee break and usually work on some additional papers. There are a lot of terms and processes concerning the immune system and also many studies being conducted geared towards learning how to best manipulate the immune system to help a wide variety of patients. So these papers are of great help to keep up with the latest ongoing research and discoveries!

After my coffee break I will perform other laboratory tasks whether it is continuing to run ELISAs, prep vaccines, or measuring out various ratios of amino acids to put in the peptide synthesizer. One day I even got a chance to go to Chapel Hill to analyze one of our samples on their TEM.

After a busy day I will wind down for the night and make sure to get a good night’s sleep for another exciting day in the lab.

Unexpected Surprises

My day in the lab I must say varies a lot day to day. Most weeks we perform several Glucose tolerance tests with our mice, so the morning is spent fasting them and getting prepped for the GTT. This involves me making our glucose that we inject either intraperitoneally or with an oral gavage. After this I label tubes that we use to collect blood samples from. Then we usually have lunch and then go to the mice house later that afternoon to complete our GTT. Then finally after that I take our samples back to the lab, centrifuge them and pipette out plasma to store in our -80 C freezers.

If we aren’t performing GTT’s that day we’re usually, removing islets, and using the Perfiusion Machine to test our islets!  The Perfiusion Machine lets us take our islets from our mice and examine their insulin secretion. The machine is interesting but involves close examination so usually we don’t leave its side.

Perifusion Machine!

In down time, I usually get to help others in the lab with prepping to use the Perfiusion machine, genotyping animals, or general mice handling activities to make them more comfortable.

 

My lab is always cold but otherwise very comfortable! The office areas of the lab are beautiful since its a converted tobacco drying warehouse.  Since we’re in the heart of downtown Durham there’s always good coffee spots nearby!

Downtown Durham!

 

How To Get Your Thumb In Shape for Summer!

10:30 – 12:30: Say hello to the lab and the lab dogs. Pipette 10µl of one sample of solid phase extract into two tubes.  Repeat ~170 times for a great thumb workout. Our challenge every day is to get a coefficient of variance below 15% between each pair of tubes, which is harder some days than others. The extract we pipette out comes from a process called Solid Phase Extraction (SPE), which we did earlier this summer. The product of SPE is a purified liquid which contains hormones from the original fecal sample and methanol. Pipetting this liquid usually takes about an hour and a half to two hours, meaning I get to chat with the other undergrads in the lab! After pipetting out the samples, I dry them in the dryer under the hood which blows compressed air into the tubes, evaporating out the methanol and leaving only the dried sample. I then add the next necessary components for the radioimmunoassay (a set of standard/control tubes, buffer, tracer, and an antibody) and let the reaction (explained in my last blog post) go for two hours. While this process may sound tedious, my lab partner, Ariel, and I have said that we feel like “actual scientists” by doing this work. Mixing different colored components of the radioimmunoassays is also more entertaining than I’d like to admit! Having a great lab who gets along well also helps, and we can be frequently overheard chatting for these two hours.

12:30-2:30: Grab lunch from West Union, pet the lab dogs (pictured below), label the set of tubes for tomorrow, and help out around the lab.

A blurry picture of a good dog (1 of 3 of the dogs in the offices)

2:30-? : Add the second antibody needed for radioimmunoassays, vortex, and place in centrifuge. Clean up the bench, put materials away, and get ready to start again tomorrow. Pet lab dogs again for good measure. (Note: The dogs do not come into the actual wet lab, Ariel will take over from there and aspirate out the supernatant from both my set and her set of tubes. The precipitate left over gets placed into a gamma counter and is read over-night.

It’s not a phase, Mom. This is who I really am.

Mother, you’ve always wanted me to become a doctor. Sorry to say it, but I kind of sort of like lab life.

I can imagine your expression right now:

new meme. disappointed mom

source: http://imgur.com/SFIK2LU
And while I’ve got your attention to the lab, let me tell you more about my average day in detail so maybe you’ll see why I love what I do.

Pre-breakfast


I like to plan everything around my meals, so my schedule will be laid out that way.Almost every morning, I’ll wake up at 6:30 to go on a run or go to the gym with my friend John Franklin.

Here’s a sweaty photo of us after a 5 mile run.

After this, we usually have a seminar or workshop scheduled through the program from 9-10:15.

Post-breakfast/Pre-lunch

After I get out of seminar/workshop and have my breakfast, I’ll head to the lab. My experiment is pretty time sensitive since I have to do screenings at a certain hour post-fertilization (hpf) of the zebrafish embryos. If I’m in the middle of an experiment, I’ll usually screen my embryos right as I get in as this marks the next 24 hpf.

Middle of an experiment
During screening, I take all of my petri dishes and look at them under the microscope. I record how many are alive, dead, have pericardial edema, yolk sac edema, bent notochord, and notes for other observations.
The screening takes about an hour and a half to two hours.

Starting an experiment
If I’m about to start an experiment, I set up breeding colonies the night before and flip the breeders and collect eggs in the morning. Then I have to wait for 6 hpf to start the dosing.
In the meantime, I’ll make some solutions for the dosing. To do this, I do a lot of math and then pipette a lot of liquids into scintillation vials to create my solutions.

Lunch
Wow my favorite time of the day has arrived!

Post-lunch

Middle of experiment
My project doesn’t require a lot of tasks, just a lot of time and waiting. After lunch, I’ll usually analyze some of the data I collected from the screening or run an assay if I’m finishing the experiment.
Assays take a lot of time and are tedious. It requires transferring embryos one by one without collecting any liquid into a well plate. Then, micropipetting solutions into the wells and run an assay. It doesn’t sound bad, but transferring embryos is hard work.

Starting an experiment
Around 3-4 pm marks 6 hpf, the time I’ll start my dosing.
I do a preliminary screen to make sure that the embryos are fertilized and healthy, and then I transfer embryos into petri dishes without collection water (again, this is tedious).
After I do that for over 200 embryos, I pour in the chemical and then I’m done for the day.

Other stuff
After I finish my experiment or have time in between stuff I have to do, I usually help others with their experiments. Most of this is running assays or doing dissections.

Pre-dinner
I usually get out of lab around 4:30, and everyday I’ll do something fun. Some things I’ll do are go to Eno River, Jordan Lake, or walk into Durham. I’ll usually do this with John Franklin and my roommate Martín.

Dinner
Wow my other favorite time of day has arrived!

Post-dinner
After dinner, I’ll usually just hang out with my friends, watch some movies, or catch up on some personal work.

It’s amazing how after I step out of the lab, I don’t have to worry about anything. During the school year, the evenings are usually the busiest. Meetings and studying take up all of my time, and now I have so much time to do things I want to do.
So Mom, this is who I want am. Sorry.