Week 5 at the Donohue Lab

Hi this is Meg and I’ll be talking about my fifth week working at the Donohue Lab at Duke. This week has been far less busy than the 2 weeks before. We still have to census every day. However, many of the plate have begun to plateau, meaning that nothing new is germinating. As a result, there are less and less plates to census every day.

In addition to censusing, Lien had to hand in a rough draft for her poster on Wednesday, and her proposal on Friday. As a result, Bri, Tarek, and Lien have been working hard analyzing Lien’s data and attempting to clean it up.

On Tuesday and Wednesday, I helped clean some of Lien’s data. Often after the data is compiled, there is a problem with the order, meaning that an earlier day has a higher number of germinants than a later day, which is impossible since we don’t remove seeds that have germinated. Therefore each successive day should have the same amount or a greater amount of germinated seeds than the previous day. In order to fix this, we attempted to modify the least amount of data as possible. This meant trying to only change one day’s data. However, a couple of times the data was just too out of order to fix. As a result, it couldn’t be used and we had to throw that data away.

Data that needs to be cleaned. Each column represents the day the data is from. The highlighted row is an example of data that needs to be cleaned since on day 3, there were 14 germinated seeds and on day 4, there were 10. Similarly later in the same data, on day 6 there were 14, and on day 7 there were 11. 

Although this week has been a lot slower than I’m used to, it’s been nice to have a bit of a break, especially since my lab manager has been letting me leave earlier than usual. However, hopefully it picks up a little bit so that I’m still fairly active in the lab.

Week 4 at the Donohue Lab

Hi this is Meg again, writing about my fourth week working in the Donohue lab at Duke University, where we are researching the different effects of water potentials on seed germination. As I described in my last post, on Monday we finished rinsing seeds from Lien’s project. Then for the rest of Monday we had to census seeds from both from my experiment and Lien’s.

Tuesday morning, we had to head out to the field site in order to weed so that other plants wouldn’t affect the growth of the experimental plants. However, the field is ridiculously overgrown, with weeds taller than I am. As a result, the weeding was pretty intensive and it took us 2 hours to clear out a tiny area. Also, because North Carolina can get extremely hot and humid by around 9 or 10 in the morning, we went out into the field at 6 am and worked until 8:30. Once we finally stopped working, I went back to my apartment to change out of my muddy and dirty clothes and then headed over to the lab. For the rest of the day I censused our experiments. After lunch though, Lien and I met with the postdoc, Tarek, to discuss the analysis of our data. Basically this ended being a 2 our graduate level statistics class, but I still successfully followed along, and was pretty interested by the end.

Inside of chamber with plates

Chamber where plates are kept

On Wednesday, luckily we didn’t have to go to the field again. However we still had to census all day. In addition, we had another 2 hour meeting about analyzing data.

Then, Thursday morning, it wasn’t required, but Lien and I volunteered to help in the field again, meaning another really long day. When we returned to the lab, we censused some more. Then just like the other days this week, we had another meeting. This one was the longest yet and it went over 2 hours and 30 minutes. By the end of the meeting, Lien and I were visibly exhausted, so Bri let us leave an hour early.

Microscope used for censusing

Sheet used to census

On Friday and throughout the weekend, we just censused during the day since we had finished data analysis and field work for the week.

Once again, this week has been exhausting. However, all this work has been pretty enjoyable and has helped me to stay occupied in the lab. As I said last week, I prefer these busy weeks to the fairly slow weeks that I had when I first began working in the lab.

Blueberry picking, lunches and final presentation - Week 5 at Duke

This is Jocelyn and I'm going to be talking about my final week of EXP. Just a
reminder, I've been working on olfaction in mice at Duke University.

My last week at Duke was a quite surreal. Neha's flight back to Durham from
California was delayed until Monday night, so on Monday I worked on my
presentation. Ting presented her paper for journal club on Oxygen Sensitivity of
mitochondrial function in rat arterial chemoreceptors. It was pretty interesting.
Tuesday and Wednesday afternoon, Neha helped me with picking the appropriate
images to show for the presentation and analysis and graphing of my data.

On Wednesday morning, the whole lab went blueberry picking! It was awesome.
People who didn't have cars got lifts from people who did at the lab at 9, and then
we all met at the blueberry farm at 9:30. Yue drove me and Sahana (another high
school student), and we got lost for at least half an hour because there was no
sign for the blueberry farm! Anyway, we got there and picked blueberries. It was
really cheap too. We stayed at the farm for a bit, and then eight of us went to the
diner for lunch. This was when I really saw how much support the lab members
had for each other and how much they appreciated some "down" time.

On Thursday, it was my "big day". I presented my data and findings to the whole
lab with Neha by my side. Hiro pointed out some mistakes I had made in defining
and the results that I presented. Neha and I talked through them and she's going
to count the positive cells again. The process is really subjective because some
may count one cell has positive but others may see it as just the background, so
she's going to figure that out for me. I brought some dunkin donuts for everyone,
and Jessica (the lab technician) brought some brownies, cookies, green tea and
chips. Hiro, Natalie, Ming-Shan had some questions and I answered them with
some hesitation but with the help from Neha, it was all good. It was sort of
intimidating at first because I was presenting to very intellectual people but they
were all very supportive. Hiro said that he wasn't going to be in the lab on Friday
so he was going to say goodbye. I told him it was truly a great experience
working in his lab and we're going to keep in touch!

On Friday, Neha and I talked about the results and the countings. Then she took
me out for lunch at Sushi Love. Ming-Shan heard about it and asked if she could
come. Just five minutes later, the whole lab was walking to the restaurant. It was
really cool. Then at five thirty, I said goodbye to everyone. It was really an
amazing experience working at Duke. To be honest, I miss it!

Here are some photos from the past five weeks:

This is one of our gels for genotyping. 

This was my Duke ID card. It gave me access to the lab.

This was my lab bench area. 

This was the microscope we used for looking at the slides in different filters. 

I used this cryotome to section paraffin blocks of tissue. 

Week 3 at the Donohue Lab

Hi this is Meg again. I just finished my third week of working in the Donohue lab at Duke University. Finally after about 2 weeks of setting of for my experiment, we were able to begin on Monday. To summarize my experiment, we will be putting seeds from different genotypes and different maternal conditions (some maternal plants were grown in hot temperatures and some were grown in cold temperatures) in dishes of different water potentials. Water potential (Ψ) helps to describe where water will move due to diffusion and more specifically osmosis; the area with the more negative water potential is where the water will move to. After the plates have been seeded, we will then census the dishes every other day to count how many have germinated. Once the all the data has been collected, hopefully we will find a threshold or base water potential (Ψ_b), which is the lowest water potential at which germination will still occur, for each of the genotypes and maternal conditions.

So this week on Monday, we started to seed both my experiment and an undergraduate’s experiment. Lien, the undergrad, is also working with different water potentials but her experiment focuses on a different aspect of water potential. However, she has a very similar setup, and therefore we have been working together on both projects. Overall, on Monday, we seeded 270 plates, 120 of those with 24 seeds, and 150 with 12 seeds, which made for a pretty tiring day.

On Tuesday we ended up seeding 50 more plates than we had originally planned, which meant that we seeded 270 plates again. Then on Wednesday, luckily it was a little less with 220 plates. On Thursday, even though it was the Fourth of July, we had to come in and finish seeding for our experiment. However, because we had done extra on Tuesday, we only had to seed 170 dishes, which meant that we were all able to leave the lab by around 1.

Buchner funnel attached
to Buchner flask

Funnel and Flask attached
to vacuum

In Lien’s experiment, the seeds are kept at in dishes with varying water potentials, just as mine are. However, after 4 days, the seeds are switched from dishes with negative water potentials to dishes with distilled water (water potential of 0). The goal of this is to see if the first treatment with varying water potentials helps the seed to progress towards germination, without actually germinating. If the negative water potentials do help the seeds progress towards germination, the amount of time it takes for the seed to germinate will decrease. In order to transfer the seeds to dishes with water, we first needed to rinse the seeds to ensure that all of the solution from the original treatment is washed off. On Friday, we began this process.

Cutting filter paper
with seeds on it

 To rinse the seeds, first we cut out the filter paper that the seeds were resting on in the plate and placed it on paper that absorbs the excess solution. Next, we placed a piece of filter paper in a Buchner funnel, which fed into a Buchner flask. The funnel is made of ceramic and has many small holes. The outside of the funnel is surrounded by a rubber stopper to create a seal. The flask has a hose barb so that a hose can connect the flask to a vacuum. Then we placed the now dry filter paper with the seeds in the funnel on top of the other filter paper. 

Rinsing seeds. The soapy-
looking water in the flask is
the dirty water that is sucked
in due to the vacuum

Next we rinsed the seeds twice with 10 ml of distilled water. Because there is a vacuum in the flask, but the paper cannot pass through the funnel, only the water that the seeds are rinsed with moves into the flask, leaving the seeds clean and sitting on dry filter paper. Finally, we placed the original filter paper in weigh boats filled with 20 ml of water, which is where they are seeded from onto the new distilled water plates. This whole process is really time consuming and stressful because the water scatters the seeds everywhere so that some are lost in the process. We couldn’t lose more than 12 of 24 seeds each time in the process or else there wouldn’t be enough seeds to complete the experiment. 

Rinsed seeds in weigh
boat, ready to be seeded

Also, because the time the seeds are placed in the dish is important to the experiment’s results, we couldn’t take any breaks until we had rinsed all the seeds for that day. As a result we had to work from 9 am to 3pm with no breaks. In addition to this, because plants don’t understand the concept of weekends, we had to work through the weekend, rinsing seeds for 6 hours straight. Finally this Monday we finished rinsing, meaning that for now on we only have to census the seeds since all seeds are in the correct plates and solutions.

Although my third week felt extremely long and stressful, and I haven’t had a day off in a while, this week was actually pretty enjoyable. In the weeks prior, seeding was pretty annoying. However, this time around, because it was my own experiment and we had been preparing so much for this week, the seeding was actually pretty exciting. I thought it was interesting that the in first 2 weeks of the lab I had just been helping everyone around me, but now everyone was actually helping me. In addition to this, the busyness of this week helped the days pass by quickly. I remember my first week in the lab the days seemed to last forever because all I really did was read articles since there wasn’t much else to do. Now that I’m a lot busier, my days feel a lot quicker and are a lot more interesting, making my overall experience a lot more enjoyable.

               

Counting cells - Week 4 at Duke

This is Jocelyn again from Duke University working on olfaction in mice. This is
my fourth week.
I thought the previous week was low key but this week topped that. The first two
days were spent genotyping new litters of pups from the animal house and other
analyses of microscope photos in ImageJ (and the help of Microsoft Excel).

For genotyping, Neha collected toe clips from the pups and we soaked the
templates in 100 micro liters of tail buffer and 10 micro liters of proteinase K
which breaks down the protein, and then put the tubes into the PCR machine. The
next day we took the tubes out and made a master mix (dNTP, forward and
reverse primers, 10x buffer and the diluted template). I ran 3 gels because there
were 27 tubes and each tube needed 4 (1 wt, 1 m, 2 wt, 2 m). 108 + 4 controls =
112 wells.

From Tuesday afternoon to Friday, Neha left for California, leaving me to code and
count positive cells in the InSitu photos. There were four folders of photos to
count. I created new excel files to record the numbers. I counted the positive cells
blindly which meant each photo had a randomized number so my results would
not be skewed. After all the counting, I recoded the photos to their genotype (WT
or M) and condition (Control or Test). The results are looking promising. Neha
said she'll take a look at them early next week.
I've started planning my presentation for the lab which I will be presenting on
Thursday before my last day on Friday. It has been such an awesome experience
working with Hiro, Neha, and the rest of the team. I wish I had photos to show
but I'll share them the last week!

- it seems like my entries are getting shorter and shorter every week.. hopefully my last entry will be more interesting!

Week 2 at the Donohue Lab

Hey this is Meg Dalrymple, and I will be writing about my second week at the Donohue Lab, which mainly focuses in the evolutionary and genetic mechanisms behind germination at Duke University

The Autoclave

Walking into the lab on Monday of my second week, I felt much more prepared and comfortable than just 7 days prior. We still had one more day of censusing left to do and so me and Lien got started on it right away and finished pretty quickly. After that, we had a lot of dirty petri dishes that needed to be sterilized before we threw them out, since they contained biological waste (the germinating seeds). So, we headed over to the autoclave, which is a big machine that sterilizes things using heat and water. There are different types of cycles that you can run, depending on what you need to be sterilized. For the trash we used a setting called “gravity” which uses heat and water, but other dry cycles only use heat. After about an hour the trash was done so we took it out of the autoclave and to the dumpster. In the afternoon, we realized that we had cut the filter paper the wrong size for our experiment. However, the new uncut filter paper that Bri had ordered hadn’t arrived yet, so we continued to cut weigh boats and punch holes in them.

The next day on Tuesday, the filter paper came. However, I busy helping the other group in the lab prepare for later in the week so I couldn’t help cut the paper. I was busy preparing plates with which the group would seed (put seeds on the plates). This meant that I counted and laid out 900 plates. Next an undergraduate, Aman, made agar to be poured into all 900 plates. As I went to lunch he started pouring. When I returned, he told me the other agar was in the autoclave and that it would be done in about 5 minutes, and then he headed off to lunch. I went up to the autoclave and got the agar so I could begin pouring. About a half hour later, Aman returned and we continued to pour. In addition to pouring agar, the plates also had to be labeled. So as we poured, we labeled and organized the plates into the different seed genotypes. After we finished pouring the agar at one point, we realized that some of the plates we not solidifying. Aman figured out that because the liquid agar had been sitting for a while, the solution had separated and the top was mainly water, and the bottom was mainly agar. Therefore, we had to redo about 200 plates and make new agar to insure that we did it right. We finally finished pouring and labeling a couple hours later.

Cut filter paper

Then on Wednesday, Lien and I began to assemble our plates. This meant that first we would sterilize the cut weigh boats with ethanol (they couldn’t be autoclaved because they would melt). Next, because we cut slits in the strip of filter paper, we were able to create a loop out of it by locking the 2 ends together, using the slits. In this loop of filter paper, the weigh boat would slide in upside-down so that the bottom of the weigh boat was facing up. Then the weigh boat and filter paper would be put in a petri dish, lying flat. On the first day, we made about 120 dishes and then helped the other group seed.

On Thursday, our only goal was to finish making the dishes. We spent all morning and some of the afternoon working on it. Eventually however, we ran out of weigh boats and had to stop. This meant that we had to order more weigh boats, and eventually will have to cut and poke holes in them. We still have around 250 dishes that we need to make. Once we were unable to make any more dishes, we decided to autoclave some distilled water because we will need it for our experiment. For the rest of the day, we read articles.

The next day we wanted to make the solutions that had different water potentials since we will be seeding our experiment on Monday on dishes with those solutions. However, we needed to first autoclave empty bottles that would hold solutions. Therefore, while we were waiting for the autoclave to finish, we helped the other group seed until lunch. After lunch we began making the solutions. In order to change the water potential of solutions, we added a powder called polyethylene glycol (PEG). PEG is actually commonly used in medicines, in chemical spills, and in toothpastes and creams. It smells a lot like glue and when mixed together with water, it creates a soapy solution. We made 5 different water potentials by weighing out really large amounts of PEG and then mixing it with water. Once we were finished, Lien and I had to go to another discussion about a paper. However, this time the paper was far easier to understand, and I had already read it earlier in the spring. Therefore the meeting was a lot less stressful than the one we had the week before.

Overall I enjoyed this week because although I’m still not doing very exciting tasks, there is more variety to them now. I’ve learned how to do many new things this week, such as autoclaving and preparing dishes, making my research more interesting and enjoyable. In addition, everyone in the lab is still really friendly and enjoyable to work with, making the overall experience a good one.

Week 3 with smells and further discussions

This is Jocelyn and I'm working on olfaction in mice at Duke University. This is
my third week here.

This week in general was very low key. For the first two days, I counted cells and
analyzed photos taken by the microscope on ImageJ. These photos were of the olfactory
epithelium. There were two versions of the photos we took. One was the nuclear
staining which stained nuclei in the tissue; the other was the cy3 staining which
is the antibody staining. Unfortunately I don't have any of those photos but
they're brilliant! Especially when you see the different colors under the
microscope.
For journal club on Monday, Jianghai presented his paper on Expression of Ectopic
Olfactory Receptors. I read the paper in advance so I understood most of what he
discussed during the meeting. Next monday, Yue is going to present about
earwax! Yue got held up by some bureaucratic stuff and only came back last week
from China. Anyway I'm excited to read the paper.

On Wednesday, we meet with different departments and first year grad students
present their research topic. This week Edward presented the effect of UV light on
egg-laying site preference in Drosophila. It was really cool! After that, the rest of
the day was pretty uneventful and I got to leave at 14:30! which was nice since I
had been staying until at least 6 on some nights the previous week.
Neha gave me some papers to read about RTP1 and RTP2 proteins. There were a
lot of terms I didn't know and since I didn't have my computer with me that day, I
asked Neha to explain them. She ended up taking half an hour explaining all of
the terms in full detail. It helped me understand our project even more too.

Yesterday we made a plan to make two clearing solutions (Scale and SeeDB) that
make the brain samples clearer to see. Scale contained urea, glycerol and triton X.
SeeDB contained different %s of fructose. We made all the calculations. Then we
made 4% Pfa which fixes the brain samples onto the slide.

(People sensitive to or against using animals in the lab, please don't read this
paragraph because you'll be disturbed. I was.)
After lunch - we were going to dissect. Neha didn't specify what we were
dissecting so when she brought in six mice to sacrifice then dissect, I was
shocked. The lab's method of euthanasia was a chamber full of CO2. We went into
the dissection lab and Neha showed me how to sacrifice the mice. She grabbed
one mouse by the tail then put it into the chamber. After two minutes or so, the
mouse was still and Neha basically dislocated the vertebrae from the rest of the
body. Then she decapitated the mice and started dissecting. At this point, I was
speechless. The room started getting really cold - I think that was just me. I told
Neha that I couldn't do it and she was really nice about it. I ended up sacrificing
one mouse but I couldn't dissect it. It was definitely a once-in-a-lifetime
experience.

Earlier in the week, Hiro (my PI) came to talk to me about what I'd be doing on the
days that Neha would be gone (next wednesday-friday). He is a great PI and so
hands on. He's been working with another summer student and he's always
around. I told him that we needed to present our projects at Science Night at the
end of fall term and he offered to make a time for me to present to the lab my last
day. I think it's going to be a good experience for me! to talk to grad students
about the research Neha and I did. It'll be good practice for Science Night too!

Week 1 at the Donohue Lab

My first day in the lab, I was the first to arrive. I had somehow overestimated my total travel time by about 30 minutes, even though it was a simple 10 minute bike ride. As a result, I showed up at 9:30 and waited in the hallway until someone showed up at 10, when I was supposed to arrive. Finally an undergraduate student, Lien, arrived and unlocked the door to let me in. She told me that she was a rising sophomore at Duke and that we would be working on the same project together. Since the lab manager who will be helping me this summer was running late due to a doctor’s appointment, Lien began to introduce me to the project. She gave me a paper that Bri, the lab manager, had given to her to read that was a good introduction to our project since it outlined the procedure. Basically in my project, we will be keeping seeds from five different genotypes, some of which were matured in hot conditions and others in cold conditions in different water potentials for 4 days. After 4 days, they will be moved to water and the amount of seeds germinated will be counted. However we aren’t starting this project until another couple of days, so until then, I am busy helping with another experiment. After I finished the paper about my project, I met with Bri, and she told me I could start censusing seeds. This basically means counting how many seeds on a dish have germinated under a microscope. It actually is harder than it sounds because the seeds are really small and often the root that breaks from the seed is clear. As a result it took me a while to get a hang of it, but by lunch I was doing alright.

Plates with dishes that needed to be censused

When I returned from lunch, Bri had to go to a lab meeting, so I continued to census, which gave me an awful headache after a while. Then at 3, Bri came out and told me that me, her, Lien, Tarek (the postdoc), and Dr. Donohue were going to meet to discuss our project. I headed into Dr. Donohue’s office and waited for the meeting to start. Basically we talked about what the point of the project is and what Dr. Donohue wants us to focus on in the next couple of days to prepare for the project. Then we planned what papers we should read and what days we would discuss them. The meeting ended around 3:45. When we came out, we discovered that one plate had yet to be censused. As a result, Bri asked me to finish it. I finished around 4 and then looked around and everyone was beginning to leave. I turned to Lien to ask if we were allowed to leave and she told me we could so I packed up my stuff and headed home for the day.

On my second day, I showed up too early again, but luckily this time Bri was already here so the lab was unlocked. I asked her if I should begin censusing again. She told me that I should and then showed me to the room with all the plates of seeds. She explained to me the system that they used to know which plates had been censused, and which still needed to be done that day. I started by grabbing one plate and initialing that I had done it. By 1, I had censused about 6 or 7 plates, which meant around 180 dishes, each with around 12 seeds on them. But luckily, between me and Lien, we were able to finish all the censusing for that day by lunchtime. As a result, when I returned from lunch, Bri told me and Lien to review some papers since we will be discussing them on Friday. I spent the rest of the day reading and taking notes.

On Wednesday, another group needed help preparing their project, so rather than census seeds, my group helped them. We had to seed, meaning that we had to put 20 seeds on Petri dishes filled with agar in a 5 by 4 grid. The seeds were extremely small, making the work quite tedious. However, it gave me the opportunity to talk and get to know people from the lab who I normally don’t work with.

Then after lunch, we finished seeding, so I began to set up for my project. This meant cutting and counting 1200 strips of filter paper that the seeds were going to rest on in the dish.

On Thursday in the morning we did more censusing and then I finished cutting and counting the strips of filter paper. After this, I reviewed a paper that we were going to discuss together on Friday. When I returned from lunch, I cut and poked holes in weigh boats for my experiment so that they would fit in dishes where the seeds were going to grow.

Censusing in the field

On Friday, I had to come to the lab an hour early because we were going to go look at the field experiments which are about a 15 minute drive from the campus. There were three different experiments that were taking place. However 2 of them were finished for the summer because seeds were no longer germinating due to the heat; they had entered secondary dormancy. One of the experiments was still going on though so we censused the seeds that had begun to bolt and marked them down on a sheet of paper. It was surprisingly tiring and by the end of the morning my knees were sore from crouching to count seeds. However, it was cool to be able to see the lab’s field experiments, even if they were all overgrown. After lunch I censused some seeds in the lab and then discussed a really confusing paper that pertains to my project. Walking into the discussion, I was nervous that I wouldn’t know anything. However, once it started, I soon realized that I actually did understand most of the paper, and that it was ok if I didn’t understand everything. This reassured me, and by the end of the meeting, I was active in the discussion and actually enjoying myself. 

Overgrown field with weather station in middle
that records weather conditions of the field

This first week has been long, and sometimes tedious with all the censusing and seeding. However, the people in the lab that I work with have been really welcoming and have so far made this experience really fun, even though I was nervous and apprehensive walking in. I’m now excited to get to know everyone better and working on my project in the upcoming weeks.  

Staining and Running Gels - Week 2

Week 2 at my lab started off with just reading at my desk. I would always
get there around 9 but Neha would usually get there at 10:30 so for an
hour and a half I just read papers, checked my emails, and did any
unfinished work from the previous day. The Matsunami lab has a journal
club where all the grad students present a paper they read on Monday and
Hiro buys us lunch. It's like when we presented the scientific papers we
read in fall term only much longer, more sophisticated and of course with a
more in-depth understanding of the topic itself. All these papers are
related to olfaction in one way or another. This week Neha presented a
paper on "Sexually Dimorphic Neurons in the Ventromedial Hypothalamus"
that govern mating in both sexes and aggression in males. I tried reading
the paper in advance to prevent any "getting lost" but eventually she lost
me in the middle of discussing the different techniques. The parts that I
understood were really interesting. Next week, Jianghai is going to present
on Ectopic Olfactory Receptors and I have yet to read the paper!
After the meeting, we did Phospho S6 staining on four slides (2 control and
2 test), Caspase-3 staining on another four slides (2 control and 2 test)
and 2 negative controls. Phospho S6 and Caspase-3 are both antibodies
that will bind to the respective proteins in the cell. The S6 protein is
phosphorylated only when mRNA is being translated so the test mice
(which were put in the two paper buckets with acetophenone) slide should
have more staining than the control mice because acetophenone is
activated. Then we added a secondary antibody that would bind to the
primary antibody (Phospho S6 and Caspase-3). We also washed the slides
with blocking solution (which is actually skim milk!) because it prevents
any non-specific binding of the antibodies. We want our secondary
antibody to bind to our specific primary antibody. With the Caspase-3
staining, the KO mice without the RTP1 and RTP2 proteins would have more
staining because casp-3 is an indicator of cell death. Neha told me that the
smell cells of the mutants die quicker so there would be more staining in
the KO mice than in the WT mice. The 2 negative controls just had the
blocking solution and no antibodies.

After the staining we put the slides in the cold room (-4 degrees). We then
took pictures of the slides on the microscope with different light filters. We
took all the photos in black and white so they're not as pretty as you would
see on the microscope. Yesterday I started counting the positive cells for
the staining, and the number of cells with nuclear staining (bisBenzimie).
Crider came to visit at around 4! and I introduced her to Neha. Neha
showed her around the lab, microscope room, and the dissection lab. She
also got to meet Hiro!
Then Crider took me and Meg out for dinner! The food was good, but I'm
not going to comment on the restaurant itself! Thanks Crider for a nice
evening! You made me think of the bike trip and, oh, how I miss it dearly!
YOBTO '12

The next day, Neha went to the animal house and clipped the mice pups'
toes to collect DNA. Thankfully I didn't have to do any of that. She came
back with 15 tubes each containing little toes - which freaked me out at
first but then we mixed tail buffer and proteinase K to each tube to degrade
the proteins. We put these tubes into the PCR machine. Then we made a
master mix that contained water, 10x buffer, 2mM dNTP, Taq polymerase,
and the forward and reverse primers of the sequence. We centrifuged the
tubes and ran gels to determine the genotypes of the pups.

On Friday, Neha transferred the pictures we took from the microscope to
her computer and I spent 4-5 hours counting the number of cells in the
neuronal layer, the number of positive cells from staining and recorded the
area of the neuronal layer. Although I don't like the computer work very
much, my work in the lab has been very balanced between hands on
experiments and data analysis on the computer.

Hope everyone else is having a good time!

Making Sense of Scents - Week 1 at Duke

I thought I was done with the visa stuff the moment I had sent in the forms to Jason, the human resource manager, but first thing I had to do Monday was to meet with Visa Services. This building was pretty far from my apartment so my dad and I took the bus. After that we went off to the International House (IHouse) for a "scholar orientation". It started pouring while we were walking there so we waited for the rain to die down under a tree. When we got there, I had to fill out some forms. Lisa came to talk to us about how to get around Duke and the Durham area as well as student activities. She gave me a whole stack of information which was really helpful. The first time she talked to me, she called me "my dear Jocelyn" which actually made me feel uncomfortable at first but I got used to it because she kept on referring to and talking to "her dear Jocelyn". It started pouring again and the thunder kicked in too. My dad and I didn't know how to get to the Duke Eye Center from here, but Lisa was so kind in offering to drive us there. She was like a "grandma-you-would-want-to-meet-in-the- woods" as PJC so refers to, although she wasn't too old. She was just really nice.

I called Hiro (my PI) from the Duke Eye Center and he said he would walk over in a bit. Next thing, we're walking to the lab just having a conversation about the summer. We walk into the building and I finally meet Jason! I still have yet to give him a present! Anyway, we have a lab meeting and one of the grad students presented a paper that she read. I realized after a minutes into the meeting that the whole lab was Asian. Not one single non-Asian. I thought that was pretty funny.

After the meeting, I was introduced to the grad student I would be working with: Neha. She showed me around the lab and I was introduced to the other lab members. I talked to Natalie, who is the only undergrad, about her research. I also talked to Mingshan about her topic of research. Then Neha brought me and another high school student to get our Duke cards. Neha also described her project to me.

For the remaining hours of my first day, I counted glomeruli in the olfactory bulb from pictures that Neha had taken with the microscope. Pretty eventful first day. Then the remainder of the week I read some review papers on the olfactory system. Our project investigates the difference in number of glomeruli in the olfactory bulb of wild type mice and knock out mice. The wild type (WT) mice have two proteins: RTP1 and RTP2. The knock out (KO) mice have neither of the proteins. Neha did 6 mice crosses and we genotyped the mice with the gel results we got. She told me that RTP1 and RTP2 genes are linked so if one mice had RTP1 the chance of it having RTP2 would be very likely.

Neha already had the DNA sequences ready for the gel so we made the mix and the gel. Each mice would have 4 columns in the gel: 1 WT, 1 M (mutant/knockout), 2 WT and 2 M. The numbers are the protein RTP1 and RTP2. She told me that they had designed the primers for the sequences so if the band is visible on the gel that means the sequence annealed to the primer. If in one mice, there are bands for 1 WT and 2 WT, that means the mice is homozygous WT, having both RTP1 and RTP2. If bands are all visible for 1 WT , 1 M, 2 WT and 2 M, then the mice would be heterozygous.

Then another experiment she did was put mouse 1 in a paper bucket as the control. She put mouse 2 in a paper bucket, then another paper bucket with a smell (acetophenone). We had four slides of the cross section of their olfactory bulb after the experiment. We did RNA in situ hybridization. For slides 2 and 7, she had a cFos probe that would bind to cFos that has DIG. For slides 3 and 8, she had a Gs probe that would bind to Gs which is present in all the cells. Slides 2 and 3 were from mouse 1 and slides 7 and 8 were from mouse 2. She also added a primary antibody which would bind to DIG. Then a secondary antibody that binds to the primary antibody, which has properties that allow for fluorescence. If the probe has cFos to bind to then there would be a lot of staining (purple in our case).

Apart from these 2 major experiments, I also got to section paraffin blocks of tissues for cross sections to put on slides. I got to use this really cool machine. I thought it was awesome when I first saw Neha use it but now that I have sectioned 4 blocks onto 20 slides of at least 10 cross sections each, it has become pretty tedious and actually somehow painful because the machine is at -20 degrees.

The lab members Mingshan, Ting, Neha, Jianghai, Jessica, Natalie and the other high school students are all very approachable. Yesterday when I was reading review articles, Jessica, the lab technician, asked me if I had plans for the weekend and I had absolutely none. That's why I'm writing this right now! But she gave me a bunch of recommendations to go to Chapel Hill, 9th street and other places in Durham. So far first week has been going well! Hope to do even more hands on work though!