Category Archives: Week 5

Lillian’s Lab Life

So far, my summer has been exciting. I’ve learned so many new things and have gotten he opportunity to talk/listen to so many amazing people. I cannot wait to see how everything turns out over these last few weeks.

Daily life in a lab is different everyday. Some days are consumed by experiments, while others are filled with data analysis.

The time at which my day starts depends on what’s going on that day. If there is an experiment that has to be done by a particular time, then I may come in early to make sure that I finish the tasks in time. Other days, I come in later than normal because of BSURF scheduled events.  I usually start my day by checking in with my mentor to see what I need to have accomplished by the end of the day (or by the end of the week). Afterwards, I’ll usually start working in the lab (unless I have to analyze data for the day). Before lunchtime, I am usually extracting cells that need that will be analyzed at a later point. Extracting cells is a much more tedious process than I initially expected but I have gotten better at it as the summer has progressed.

Lunchtime is always a great time. Sometimes I’ll get lunch with my mentor and some of the other people in the lab. There is another undergraduate in the lab (who is also named Lillian) and we like to get lunch together if we are both free at the same time. There are also times when I have to get a quick lunch, especially if I am in the middle of an experiment, so I will go by myself.

After lunch, if I extracted cells in the morning, then my afternoon is consumed by the staining of those cells. If they have to be analyzed immediately, then those cells will also be taken to the flow cytometer. Sometimes I will also have to count cells, a task I did a lot of during my first week in the lab. It used to take me a very long time to count one sample but now I am much better. One of my favorite things to do in the afternoon is genotyping. I am completely fascinated by the process of gel electrophoresis, which makes the process of preparing and running the DNA samples exciting. In the afternoon, I may also prepare solutions for future experiments or watch the experiments being done by other members in the lab.

Overall, day to day life in a lab is always full of something new.

 

A Day in the Heitman Lab!

I have loved every moment of of my research project this summer. Although its been challenging adjusting to new terminology, vocabulary, and other hands on task, I could not ask for a better opportunity. My days in the lab have been pretty consistent for the most part, with few variations from the norm.

First I arrive around 10:30 where I go straight to my notebook to make sure I have documented everything I have done from previous days. Once I have done this I have a talk with my mentor, Ci, to understand what my next steps are in the project. Once we have had our discussion and I am sure that I understand what we are doing and the reasons why I begin setting up and preparing to run the experiment (most days this would be a PCR overlap). I write in my notebook every reagent that I will be using in order to ensure that I do not make any mistakes. Once I have completed this task, I begin conducting the experiment. I transfer the correct amounts of each primer and DNA template based on my master mix into PCR tubes.

Once I am sure that the correct reagents are in each tube I then place these tubes into a thermocycler (also known as a PCR machine). I program the machine based on the protocol of the polymerase that I am using. Once I confirm the time and temperature of each step, I start the machine to allow it to do its task of amplifying and constructing the DNA strands.

After the machine has finished amplifying the DNA strands, I take each PCR product and prepare it for gel electrophoresis. I do this by adding 5 micro-liters of 10x dye to each tube. Once this is finished I transfer each reagent into a separate well in the gel and run the gel in the electrophoresis machine.

Hopefully after doing this process I have successfully constructed and amplified the DNA strands that I need to continue my project. To confirm whether or not I have done this, I take a look at the gel using Ultraviolet light and see whether the position of the bands compared to the ladder matches my predicted outcome. If not, then I have to go back to the drawing board to see what went wrong. If so, then I will purify each PCR product that came out successfully and then continue on to the next step in the project. And that’s my day!

Collections, Dissections, ChIP, Analysis, Repeat

Like I have mentioned in my previous blog posts, my experiment is based around seeing what genes effect sexual behavior in the brains of flies. Depending on what gene (fruitless, doublesex 1 or 2, or ChAT) and what epigenetic marker (H3K27ac) or a proxy of transcription (RNA Polymerase II) we are focusing on, my day in the lab may be different.

For example, if we are just starting to look at one of the genes and epigenetic markers listed above, I will either collect adult male flies in groups of 30 or isolate fly pupa into their own bottles (1 pupa to one bottle). These collections set up group housed or isolated flies for my experiment.

Those flies are kept in their environments for about 5 days, so across that span I can collect more flies or start dissecting flies from previous collections. For dissections, I take the head off of the body of the fly and remove the mouth.

After dissections, ChIP protocol is followed to isolate the DNA that is related to the epigenetic marker we’re looking at. Following ChIP, we use computer analysis to quantify the enrichment of the respective protein or mark that the flies received from their respective environments at the promoters for each respective gene of interest.

This cycle is essentially repeated over and over again, so whatever stage of the cycle I am at is what I do during a day at lab. If I had to choose, collecting and dissections are my favorite parts!

Stage 5: So I can’t copy/paste my lab notebook here?

With a full day of benchwork planned, I try and wake up at 5 am, but the nauseating breach of sunlight through the blinds often send me back under the covers for another 5 minutes. Or maybe 50. Given the former, I head to the gym briefly, shower, and hustle it to lab to consolidate the day’s different goals into a precise schedule and starting it before the BSURF meeting. Given the latter, well, might as well round 5:50 am to 6 am, which is essentially too late for a whole workout routine, so might as well conserve sleep until 7 am, but I don’t actually need breakfast, so 7:45 am works, but construction is bothersome, and therefore, I hope my 8:40 am alarm catches me in time for the BSURF meeting…Maybe 8:41?

After BSURF, I drift back to the Bryan Research Building at around 10:20 am. Christine, the only other undergraduate in the Colton lab, is hard at work preparing and reviewing her PCR data on ground squirrel brain tissue but doesn’t hesitate to glance up and greet anyone who walks into the lab. Joan, a technician, is mysteriously working on the other side of the bench, somehow switching constantly between benchwork, lab management, and mice care. Three coffees, some slight joking, and one lively Dr. Colton visit later, the day officially starts. What I do depends on where I am on the experiment. Some days are laborious: passage the cells, treat the experimental lines, prepare buffers, read articles, prepare the next experiment. Other days are lazy, and I might sneak in a nap for my typically brisk lunch break, although 2 more hits of coffee will suffice before bugging Huifang and Stuart, another two techs in the lab, for guidance or just because. They’re really cool, and also somehow do the things that take me a day in an hour. And then some.

If I haven’t clumsily destroyed something (it’s only happened once or thrice), then the day is done! Which is usually around 5 pm because although precise, efficiency evades me on the best of days. *looks nervously over at all 8 BCAs I have done sans knowledge of the glorious multi-channel pipette* *furious glance at Western blot gel that blew up and had to be meticulously jigsawed back together* *desperate groans as water/bead bath takes an hour heating up a singular media for a one-step protocol* *flies into hysterics at all the reagents that have conveniently run out right before the last sample* Although sometimes, I just stay in lab a few extra hours just to peek at some IHC slides Angela, another tech specialized in churning out hundreds of these things (it’s really quite amazing), has prepared under a microscope for different stained proteins or cell types. Past 4 pm, most everyone has left besides Stuart or Huifang, and so, I pester them a bit more because they are awesome, and try and get the next day worked out. After 6 pm, everyone is most definitely gone, and if not, woe to them because their only remaining company would be me! I spend around 30 minutes to an hour completing my lab notebook and reviewing calculations or data from the day, and if I am still awake, I glance through some more articles to see what I want to do next. All this makes me feel productive, which is comforting because as soon as I get back to my room, I eat and sleep. Rinse. And repeat. I cannot imagine fitting this into a semester of classes and extracurriculars, but nights are long and I really enjoy it here so I’ll make it work. It better work. Usually, something works.

Weekly Highlights

“This is Dr. Colton’s place!”-Joan Wilson, proceeding to turn into a forest
“What”-Dang Nguyen as the next half mile is spent driving through straight gravel to get atop of a hill

“I prepared the cells!”-Dang Nguyen
“What? You prepare the cells the day before transfection.”-Huifang Dai
“Oh. Uh.”-Dang Nguyen ruining his weekend plans right before heading home on a Friday afternoon

“Now we’re finally done!”-Huifang Dai at 6:11 pm after a long day of Westerns
“Huzzah!”-Dang Nguyen as he turns around to head home just a tad too violently, merrily spilling the last drops of antibody dilution they had just finished

“We’re here!”-Joan Wilson breaking into Dr. Colton’s backdoor and walking straight in

“Happy birthday guys!”-Dr. Carol Colton with a birthday cake
“All [f]our birthdays were weeks ago”-Stuart Sundseth
“…It’s America’s birthday”-Dr. Carol Colton

All In a Day’s Work

Although every day in the lab is slightly different, there are some constants that exist. I usually get to lab and do germination checks for about two hours in the morning. I have my petri dishes where we put down 20 seeds in each dish, and I have to go through different cohorts each day and record the number of seeds in each dish that have germinated. Afterwards, I usually analyze the germination data from previous days using different statistical methods on R, or practice making different types of graphs. Sometimes I meet with my mentor to discuss different methods or if my interpretations of the results are valid. This usually brings us to a late lunch around 2, where I either nap for 20 minutes or quickly eat chicken nuggets at the Bryan center.

 

After lunch, I do odd jobs around the lab for my lab manager for an hour. I usually help the other grad students in the lab in this time. These jobs include performing crosses between different ecotypes of Arabidopsis. Then it is time to check the plants that have been planted in the phytotron. This includes recording which plants have bolted, flowered etc. Occasionally we will water and fertilize the plants. We put sleeves/plastic cones on the plants that have started to develop so we can collect their seeds. We also make sure that the plants aren’t touching each other. After these checks, sometimes there are more germination checks to do, so I will do the final checks and leave.

A day in the life

When I first walk into lab, I check my email to see if there are any updates from my mentor on my project. Then I prepare PCR to test the primers for Shank1 and Dlg4, as I am finding that I have to adjust the master mix to minimize dimers that appear during this PCR, Once I stick that day’s PCR in the thermocycler, I then run a gel for PCR that I did overnight. After doing that, I consolt with my mentor about what the gel means, and what further alterations to the mastermix I should make. After this, I take my lunch break where I read protocols and the literature. After lunch, I spend a few more hours experimenting with optimal conditions for Shank1 and Dlg4 primers in preparation for pyrosequencing.

Once I am done optimizing these primers, it will be time to pyrosequence to test for methylation, and then use RT-PCR to test for gene expression.

Life in the Lab

I usually get to the lab quite early, in order to get a head start on my experiments before going to the usual BSURF meeting. Many of the experiments we run, such as Western Blots and RT-PCRs, require a lot of preparation and busy work (including a ridiculous amount of pipetting). A procedure can take up most of a day, if not multiple days, and you cannot simply stop and start at whatever point you like. There are set points in the procedure that you have to reach before calling it a day, and getting started early is therefore crucial, if you want to avoid having to stay late. For example, the membrane in a Western Blot has to be washed overnight in primary antibody, so that it can be developed the following day. Failing to set up a primary antibody wash before leaving would mean waiting a whole additional day for your results. For other procedures, the intermediates we work with are easily degraded, and must be converted to something else before being stored. RNA is particularly fragile, and after being isolated from cells must quickly be converted to cDNA. Therefore, you must perform the necessary reverse transcription reaction before finishing up for the day.

Another regular task is cell maintenance. After several days of growth, cells become overly “confluent” – crowded on their plate – and their growth media becomes depleted. In order to keep our cancer cell lines growing and healthy, we have to “passage” them every few days. This involves taking a fraction of the cells that were growing in culture and transferring them to a fresh plate with new growth media. A substantial amount of work goes into simply ensuring that you have cells to perform experiments on, let alone performing the experiments themselves!

This past week has been particularly busy, as I have been doing a time course of GNF5 (ABL kinase inhibitor) treatment – essentially, I treat cells with the drug at a number of different time points over a period of 72 hours. This has involved coming to lab several times a day, often at odd hours, ranging from 7AM to 11PM. The LSRC is a somewhat eerie building that late at night!

An entire experiment, from plating cells out for a drug treatment to producing presentable data by developing your Western Blot or analyzing your RT-PCR, takes at least several days. It is therefore important to enjoy the procedures themselves, and not only be in it for the results  – otherwise you are likely to find yourself bored and frustrated most of the time.

A Day in the Lab

In my lab, not every day is the same. Depending on what is going on with our project, some days may be busy days, and some days may be “waiting” days, when we are waiting for our experiments to run. As I stated in my chalk talk, the main thing my mentor and I are working on is isolating exosomes and looking at exosomal biomarkers.

When I walk into lab, I ask my mentor what the plan is for the day and we discuss any bench work that needs to be done. These past few weeks, my mentor has been running trial experiments to isolate the exosomes (exosomes are kind of hard to isolate) so we can send them off to another lab for further analysis on the proteomics and RNA sequencing. As a result, on any given day, my mentor could be running an ultracentrifugation, a  bicinchoninic acid assay, doing a western blot, or passaging the cell lines for the project, and I just try to assist wherever I can.  On days where we are waiting for one of these experiments to conclude (i.e. ultracentrifugation  or western blots), we usually try to find someone else in the lab who is working on something that I want to learn about. For example, last Friday I ran my first PCR for one of my lab member’s project, so that was pretty exciting! Overall, I will say that I have learned a lot and I really admire every one of my lab members and the projects they are working on. I definitely wish I could contribute more to the lab, but I guess with time comes experience!

Episode 5- A Day in the Life (in the Lab)

For me in the Wray lab, everyday is pretty close to the same (so long as there are sea urchins in stock). As soon as I get to the lab, I check on the sea urchins I injected the day before if I did injections. Then, I check the dry erase board to see if there’s anyone planning to spawn an urchin before 1 since any time after that is too late. If there isn’t anything written on the board, I go ahead and make the injection mixtures I’m going to be doing that day and set them on ice. Then, I grab the urchin tray and bring it to the urchin room.

Once I’ve spawned some eggs, I have to wash them several times to remove the jelly so the eggs don’t stick together and dirty the water. While waiting for the eggs to settle during the wash, I set up what I need to row the eggs on some plates. After the wash,  put the eggs in a glass dish, swirl them into the center, and place them under a microscope to begin collecting them.

Using a mouth pipette, I suck up some eggs and then blow them out gently onto a plate filled with sea water and PABA (Para-Aminobenzoic Acid) which keeps the eggs soft enough to inject. If done right, the eggs stick to the bottom and don’t roll around much from where they were placed. Then, I add some sperm to each plate and swirl them around to make sure the eggs get fertilized. Now, they’re ready for injection.

To set up for the injection, I take a needle and place it into a nozzle and place a plate on the microscope stage. After a minute or two of adjusting the needle and stage positions, I can see the needle and inject the fertilized eggs. Once injection is finished, all the plates are placed in a plastic case and into an incubator to develop.

Four to five hours later, I can take the embryos out of the incubator and begin transferring them to a 12 well plate. I use the mouth pipette again to select the glowing embryos since those were the ones injected and I move them to different plates depending on the treatment and how brightly they are glowing. Afterwards, all the plates go back into the incubator to develop more overnight. The embryos have to be transferred because spending too much time in the PABA makes them sick and develop poorly.

The following morning, I check the results and see how many of the embryos/larvae have developed and if there appear to be any significant differences in the way the injected versus control animals have developed. After that, I wash and throw out the plates and begin another batch of injections.

Week 5 – Fecal Samples and Iodine

As I mentioned in my past post about my project, I am determining the thyroid concentration in fecal samples of a wild population of male baboons using T3 radioimmunoassay (RIA).

First, here’s a bit more information about how RIAs work: In a T3 RIA, an unknown amount of T3 thyroid hormone competes with a known amount of radioactively labeled T3 for binding sites on an antibody-coated tube. The antibody has equal affinity to the radioactive T3 tracer solution (in which the T3 is labeled with 125I) and the T3 present in the fecal samples. Once the T3 in the fecal sample competes with the T3 tracer solution for the binding sites, the antibody-coated tubes can be placed in a gamma counter that will determine radioactivity of the sample, which is measured in counts per minute (cpm). Seven standards (A-F and B ½) that contain different, known concentrations of T3 are analyzed each time a RIA is performed so that a standard curve can be generated. Therefore, the radioactivity of the fecal samples can be compared to the standards, and the concentration of T3 can be extrapolated from the curve.

After five weeks of working on my research project, I have performed 22 RIAs on about 1,500 fecal samples. Since the results of my project heavily rely on how many samples my mentor and I analyze, so far my days in the lab have followed a fairly predictable routine.

When I arrive in the morning I first collect the pre-selected 68 samples sitting in the freezer and prepare them for analysis the following day. To prepare them, I warm the samples to about 37°C in a water bath, vortex them for 10 seconds (more often than not there are solid components at the bottom of the test tube and we need those to be evenly distributed throughout the solution), transfer 2.5 mL of each sample into test tubes, and then dry them in the evap-o-rack. All the steps leading up to the evap-o-rack can take about 1.5 to 2 hours to complete. While the amount of time it takes for the samples to dry can vary, once the samples start drying then the morning phase of my day is over and onward I progress to the next stage of my day: performing the RIAs. (An important note: once the samples are completely drying sometime during the afternoon, I store them in the freezer to be analyzed the following day.)

In the second, longer part of my day, I retrieve the dry samples prepared the prior day and add 250 μl of Standard A (which contains no T3) to every test tube. I then vortex all the samples for 30 seconds, transfer them into Eppendorf tubes, and obtain the antibody-coated tubes. 100 μl of each standard, three controls, one pool, and the samples are added to two tubes as outlined below, ultimately resulting in 160 tubes that will be put in the gamma counter. But before I reach that step, I add 1 ml of the T3 tracer to all the tubes, vortex them for 10 seconds, incubate the samples for 1 hour at 37°C, aspirate the tubes, add 3 ml of deionized water to them all, then aspirate again. After the tubes are aspirated for the second time, I place them into the gamma counter and voilà: my day in the lab is over.

Before I leave, I like to label the test tubes and Eppendorf tubes to prepare for the next time I’m in the lab, which helps me save some time during the process. In the first couple of weeks, I often would end up leaving lab after 7:00. By week 4, I got use to the procedure and became more efficient, which helped me settle in a regular schedule of work, have a snack, work, have lunch during the hour wait, then finish up for the day and leave around 6:00. The only exception to this flow is on Mondays when I join my lab for the weekly lunch meetings. I may end up leaving the lab after 7:00 on Mondays, but it’s well worth it to spend the time with members of the Alberts lab and my mentor’s two dogs: Cyclone and Twister.

Life in the lab

For me, there are things that I do on a daily basis, but there are also things that I do that depend on how a previous day’s work turned out.

Most days, I arrive at the lab I check in with my mentor Brittany and plan out the day’s work.  Then we usually check on our cells to see if we need to split them, how to split them (1:10, 1:20, etcetera), or if the cells just need a media change.

Once we make a plan I get to work. On a given day, I might be making gels, and running gels, to get ready to develop a western blot the next day.  I might also be collecting cells, and lysing the cells to either do an Immunoprecipitation (IP) or a normal western blot.  Additionally, I will sometimes treat the cells, or transfect the cells in order to delete a certain gene or express a construct so that I can see what is happening to expression levels when I treat my cells.  Finally, I may also step away from the cells and attempt to do a mutagenesis reaction.  In order to do this, I will first set up the mutagenesis reaction (it is similar to a PCR reaction but with a different primer), and run the mutagenesis reaction.  The next day (day 2), I will do a DPN1 to get rid of the original template (so that you have your mutated DNA).  Then I will transform the new DNA into bacteria, and plate the bacteria.  The next day(day 3), I will collect various colonies and grow them out.  Then on day 4, I will isolate the DNA from the bacteria and send it off for sequencing.  On day 5, I will receive the sequence and go from there.

Some days, the science does not go as you hoped it will and some experiments have to be done again, some might have to be changed, or you might have to stop the experiment altogether.  All in all, the days in the lab in my experience is slightly different each day and builds upon the previous day.  Each day, I learn something new and it has been a great experience.  I am learning to be patient and realize it is okay if things don’t go as well as you hoped… more on this on next weeks blog where I will talk about the highs and lows of my project.

 

 

What do I do?

Through the past couple of weeks my routine in lab has kind of been all over the place. Depending on the goal for the day, I alternate between the third, fourth, and ultimately the fifth floor (where my lab is located) of the French Family Science Center.

So why does my daily routine potentially have me going all over the place?

I always start my day in the fifth floor, leaving my backpack and checking what I have to do for the day. Sometimes I will have to check in with my grad students to see if they have something they want me to do for them. but usually, I will have some kind of plan before-hand for what I could be doing. If it’s a protein purification, I get started immediately on thawing the pellets in ice (from the fourth floor) and preparing my buffers. Protein purifications generally take 3 days so, when I need to start them, I will come into lab at 8 am in order for the sample to lyse, clean, and spin down to be injected into the chromatography system at a reasonable time. (Because spinning down the sample takes such a long time, I usually will load the sample on the second day). If it’s a protein expression, I’ll come into lab at around 10am, start making the medium, and then deliver it to the fourth floor to be autoclaved. After being autoclaved, I inject the sample and wait for incubation, finally harvesting them once incubation is done.

While I wait for incubating, centrifuging, or autoclaving (I’m usually waiting for one of these things) I’ll be at my desk area reading up on papers that I’ve been given or surfing the internet. Sometimes I’ll go on a walk. (Recently, though, I’ve been working on some image analysis for one of my grad students) Once my waiting is done, I will usually be at the bench finishing up preparation. Sometimes, I will need to set up a chromatography system or gel running in the background.

So far, though, I’ve only covered the fourth and fifth floors. Where does the third floor come in? Well, the Nano ITC I’m using is in the Hargrove Lab on the third floor so throughout my day, I go back and forth cleaning the ITC and then loading samples to run and doing my work in lab on the fifth floor. I’ll usually run the ITC while I am doing other experiments and check in on it after an hour and a half.

My daily routine varies depending on what I’m doing. I rotate between floors, wait a lot, and run different kinds of experiments. Overall, I love what I’m doing and working in lab.

My favorite Chalk Talk from Last Week

I really enjoyed all of the chalk talks from last week. To be frank I felt a little proud of everyone because from what I could understand everyone had fairly complicated subjects and projects. To see that everyone could effectively explain what their projects were about and what they were doing was impressive despite out lack of experience. In addition, I thought everyone had demonstrated effective public speaking even though I noticed some were nervous (I was nervous, too). However, I really enjoyed Christine’s chalk talk because it was so different from everyone else’s.

Most if not all of us are working at the microbe or molecular level but Christine’s project has to do with quanifiying the amount of stress disctributed amongst different ranks amongst baboons. When I was first listenin to the premise of the study, I was first thinking that it was obvious that the alpha and lower-ranking baboons would have the most stress but then I realized that this was assumption based on bias and observations. Christine’s work is actually quantifies the stress by measuring the amount of stress hormones excreted by the animals. I also liked that the types of stress expressed by the varying ranking baboons were identified. Alpha baboons were marked with energetic stress, having to defend or fight for their position in the group. Low-ranking baboons were marked with psychological stress, meaning their stress originated mainly from their inability to access resources and isolation due to their percieved weakness. I guess I enjoyed her chalk talk the most because it involved a combination of behavioral science and molecular biology. Moreover, (she stressed that this was a stretch)I found it interesting about the implications that this project has on Homo sapiens. I am primarlily interested in microbiology/infectious disease but I do have a keen interest in society and how socioeconomic status affects people in an abundance of ways because the two topics interconnect in several ways.

Three Days in the Lab!

So far through this summer, I have discovered that the stereotypic perception of the routines of a researcher is mostly false. Each day there is usually a new challenge, concept, or method to try, creating an experience very much different than the monotonous routine everybody is seemingly attaches to research. It’s true that a simple task of pipetting or waiting for a culture to grow may seem repetitive, but all these things ultimately build up to contributing towards a larger goal which makes even a daily routine exciting. As I move further into my project, each day is new as there is a new step to be taken in ultimately solving a structure for my protein of interest. The things done in the lab are often determined by the results received the day before, as I have had a wide range of different days in the lab. However, here is a slight look into a possible day in the lab.

If I was just starting a new experiment or starting from the very beginning to express a protein and eventually solve a structure, I usually start off with checking my bacteria cultures from the night before. After checking the cell density of these cultures, most times I dilute them and wait till they grow toward the preferred density for the experiments of the day. While that is happening, depending on the day, I will either prepare needed solutions or samples needed for the rest of the day. After the bacteria has grown to preferred density, I usually will conduct an inducing experiment and attempt to express the protein of interest in different conditions. Often times there are time conditions involved in these different conditions so some of the day does consist of waiting. After waiting (this could be actually be done the next morning, depending of the conditions), I run an electrophoresis gel to see the results of the conditions. Analyzing these results will give me indication for what my next few days will look like. If there is significant protein expression, the next few days will consist of creating large cultures of the same strain of bacteria with the protein of interest in order to attempt a purification process. The purification process is generally consists of lysing the cells and running a column to ultimately elute the cells. If not, then it is back to the drawing board, trying new strains or methods in order to express the protein needed. I would create new cultures and grow them overnight to once again begin this process all over again.

Often times it seems like my “day” is actually three days in total as these three days is what it takes to usually complete a cycle of experiments (growth, expression, gel/purification). With results, my following day would be quite different than the preparation days. In addition, because I am now helping with a project analyzing the protein MprA, my days now also consist of ITC and other purification methods. The exact details of a day are often driven by the results of my previous experiments, leaving every day up in the air and adding an element of excitement.

 

Till next time,

Luke Sang

A Day in the Lab

I normally start my day at the lab habituating mice to a head-fixation device connected to a treadmill. Mice that are considered habituated will groom and run (and sometimes vocalize a little bit spontaneously) instead of displaying freezing behavior. Before placing them back to their home cages, I usually give the mice some sweet treats for having put up with the treadmill training that day. The goal of this habituation is to get the head-fixed mice to vocalize. Since male mice vocalize most reliably during courtship, I run behavior sessions occasionally during or after treadmill to give the males opportunities to spend time with females.

Depending on the exact content of mice training, I might do histology during or after the habituation sessions. Sometimes, the afternoon is spent learning or performing surgeries (which I’m still very slow at…).

While everyone in the lab has a busy schedule and tries the best to be the most productive every day, one lesson I learned so far is that you can never force behaviors to happen naturally. If the mice refuse to vocalize…mind-blowingly, they just don’t vocalize. If the mice dislike the treadmill…well, I haven’t found the magic wand yet to make them relaxed right away. Working in the lab has been an incredible learning experience beyond just the technical aspect. I’m learning to be patient and flexible, to troubleshoot, and to look positively at the reality that things don’t always work! 🙂

A Day in the Lab with Claire

A normal day in the lab usually begins with checking on my 293T cell lines to see if they are confluent enough to passage to a new plate. If the cells are 80-90% confluent, then I will thaw my media and trypsin, prepare the cell culture hood and then passage 10% of my cells to a new plate so that they don’t become overconfluent and die. I usually need to split my cell lines every other day.

After I do this, sometimes I will have a mini prep or a midi prep of bacteria with my designed gRNA plasmids culturing in the incubator overnight, so I will take that out of the 37° shaker. I will begin the protocol by centrifuging the tubes with my transformed bacteria for 10 minutes, so during this time I check in with my mentor. We will go over the procedures I will need to do that day and why they are necessary for my project. This is usually a good time for me to ask questions about the various protocols for the day’s tasks and be sure I understand each step. After this check-in, I carry on with the mini prep, or whatever procedure I need to complete first for the day.

By the time I am done with the day’s first assignment, it is usually time for lunch. Some days, the lab will all go out to lunch or someone will order pizza for everyone. These are the best days because I get to hear about the cutting-edge research they do and also get to know them outside of the lab setting. After lunch, I will complete the rest of my tasks, whether that includes transfecting cells, changing media, harvesting cells, PCR, restriction digests, surveyors or designing new guides for my project.

Before I leave for the day, I will transform one of my plasmids into competent cells or start a mini prep from bacterial plates that I have already prepared. This is done at the end of the day so that the bacteria can grow overnight. Overall, the lab is a relaxed, collaborative environment where I don’t feel afraid to ask questions.

Slave to my experiments AND to my urchins


“Does anyone else need eggs?” “Why is this not working?” “Should I use the other set of primers?” Questions, questions, and more questions. These are the phrases that indicate a lot of the things I am doing in lab. Performing experiments, analyzing results, then troubleshooting problems that occur.

My average day consists of me arriving to lab at 8AM to thaw solutions, start PCR reactions (since they take up to 2.5 hours to run), or check on experiments or embryos that were going or incubating overnight. After coming back from faculty talks or group meetings, I usually do the rest of my experiments which may include in situ hybridization experiments, gel electrophoresis and gel extractions to purify DNA, and drug treatments.

In situ hybridization experiments take 3 days and consist of washing embryos in a multitude of solutions. Gel electrophoresis requires me to run a gel to separate out DNA by size. Once that happens, I determine if the DNA is purified or not by checking the number of bands. If there are multiple bands, I need to purify the DNA through gel extraction and run the gel again until I end up with one single band. Drug treatments require more planning because of the timing. I usually spawn and fertilize eggs of a sea urchin and I will come in at different times (sometimes as late as midnight) to change the artificial sea water or put in a drug to inhibit signaling molecules. I would then have to fix the embryos and store them until I perform in situ hybridization experiments.

Aside from doing wet lab experiments, I also need to take and process images on a microscope and computer. This requires me to be in a dark room for a few hours and burn my eyes out looking at a screen. Luckily I am able to listen to music while doing so. Once I have the images, I need to process them so they become .jpg files.

Sometimes other things happen. For example, a pleasant surprise that happened this last Wednesday was that my mentor, Ray, baked cupcakes for my birthday.

All in all, my lab days are jam packed with experiments which I am super thankful for because of all the experience I am getting working first-hand on a research project. To end this weeks post, I want to highlight Dr. Grunwald’s prediction that we (research fellows) are slaves to our experiments. In my case, I have become a slave to my urchins.

My birthday cupcake baked by the one and only Ray Allen!

Week 5 – Chalk talks – pick one and reflect on that talk

Due Sunday, 8 July, 5 pm