Rutgers Ware Lab - Week 3

Hi everyone, this is Michael and I've been working in Ware Lab at Rutgers. We focus on studying the body and shape of dragonflies and damselflies.

This week started off me completing everything needed to be done for sequencing. The samples were submitted on Tuesday, and I got the sequence files (files used for analyzing dna on computer) back on Wednesday. While I was waiting, I scanned more wings for Will, and aligned more dna files.

On Wednesday, my professor guided me what to do with the raw sequence files, which involves playing with a handful of programs such as sequencher, clustal, mesquite, etc. Afterwards, I transformed those files into nexus files, ready to be analyzed and compared with gene database. At the end, I combined the genes coding for the same type of dna (such as 16s, 28s), and used the programs garli and figtree to produce a phylogenetic tree.

This is how the final image of a
wing scan looks like; the letters at the
bottom are the code for this sample.

On Thursday, a friend of my professor had an emergency at her lab in NJIT, and they picked me to assist her. She received more bee samples than she had expected, and she had to organize them into an excel file before Saturday. This involvd putting the code and information of each sample into rolls in excel.

The process of scanning wings. The pair of
wings are placed at the top left corner of a printer

Rutgers Ware Lab - Week 2

Hi everyone, this is Michael and I've been working in Ware Lab at Rutgers. We focus on studying the body and shape of dragonflies and damselflies.

After a week of presentations in Germany, my professor and some lab members assigned me to run PCRs for about 50 samples. Since I had to pipette chemicals into vials for PCR one by one prior to running the PCR, the process took almost the whole day. After PCR was done, however, I used a pipette with 8 slots for gel electrophoresis, which probably saved me about an hour of work.

On Wednesday, Dominic, the third member of the lab, returned from Guiana with a lot of samples. One of them is a complete massive beetle he found in his house (see image below). Along with them were bees, ants, all sorts of interesting insects.

On thursday, Will introduced me to his project: he is designing a program that can identify Odonata species just by matching images of wings from those in a large database. I decided to take part in his project. The work basically involves scanning wings in a way much like how you scan papers, except they fly away extremely easily, so I had to perform every action gently to avoid messing up the scanning process.

Dominic's beetle
This is about the size of my palm. Dominic even
says that "it's like a toy", since you can move
its wings and claws freely.

Pipette with eight slots: without this, those samples would
take hours of work jsut to run a gel electrophoresis.

Tissue Morphdynamics Lab: Week 7-8

Hi, it's Danny again from Dr. Nelson's Tissue Morphodynamics Lab and its been about a week or so since my last update. Most of my time has largely been devoted to working on lung morphogenesis or essentially lung development, but also I got introduced to a new and exciting topic regarding divisional axes in tissue. Although the two topics don't necessary overlap in terms of research, the work I did the previous year at a UCLA Head and Neck Cancer Lab is very similar in terms of techniques but very different in terms of direction.

Sriram's project focuses on the effects of endogenous stress and motility in cell division. In previous experiments, it was shown that when two cells are attaches at opposite poles of another cell, they tend to create a polarity and divide along a parallel axis. Additionally, it is known that if there is no endogenous stress on the walls of a cell, the cell divides in an unpredictable manner. The objective of Sriram's graduate work is to determine how endogenous stress and cell motility affect cells' divisional pattern on 250 to 500 micron squares.

The process in create a small micron square and treat it with the necessary drugs and cells is a rather long but straightforward. Using soft lithography one can etch patterns into a film that can be used as a base for future experiments. Lithography is useful because it can create any pattern or shape but the pattern that is desired for this particular experiment is 250 micron to 500 micron squares. An essential material of the experiment is Polydimethylsiloxane (PDMS) masters, which is made by mixing cross-linkers to PDMS in a 1:10 ratio and vacuuming out an air bubbles. PDMS masters can then be applied to the lithography film which will result in a negative copy of the film. Then the negative PDMS masters stamp is coated with silane, a substance that prevents PDMS from sticking to itself. After the silane has dried, the PDMS masters negative can be used as a stamp and reused multiple times, until the quality of the stamps produced is subpar. PDMS masters is applied to a negative base and cultured in a thermo-regulator at about 60°C for 3 to 4 hours. As the PDMS masters is being cultured, one can spin down cover slips with PDMS masters on it. Once fours hours has passed, the positive copy is removed from the stamp, which is then cut into little squares using a razor. The PDMS masters squares are then treated with fibronectin protein and then stamped onto the PDMS cover slips. These cover slips are then treated with mammalian cells with a variety of drugs and analyzed using a con-focal microscope over 12 hours.

The machine used to spin down PDMS on cover slips
in order to create an even layer.

PDMS and cross-linkers (right)
Vacuum used to remove air bubbles (left)

I was supposed to attempt to make a cover slip treated with cells this week, but the fume hoods in the culture room were replaced due to ventilation issues. Sriram told me that the insides of new fume hoods are usually quite dirty and suggested that we wait some time before culturing and analyzing new cells. The past two weeks have been a mixture of chicken embryo dissections and imaging lungs as well as a lot of Imaris and image analysis.

PDMS masters stamps that will eventually be covered with fibronectin proteins.  

The next week is going to consist of micro-dissecting about 4 lungs in the morning and 4 lungs in the afternoon for day 4, day 5, day 6, and day 7 chicken lungs in order to start preparing for my poster and presentation. I will also hopefully attempt to go through the aforementioned process with the help of Sriram, if the fume hoods are restocked with equipment by then.

In terms of life outside of the lab, I've been working on my senior thesis paper over the summer and started on the dreaded summer homework. Josh has just left his lab, so if he reads this, hopefully he got back home safely. It's been a great 8 weeks so far at the lab and I've learned so much and met so many great people.

On a side note, today is Friday and I just got a new assignment to start going back through a set of data and count the number of cells in the data that has over 110 cells at around 11 hours. I've been counting cells for the past two hours because I would really dislike doing this on a Monday morning, so if you need anyone to count up to 150 in 2's as quickly as possible; I'm your guy. 

Week two and three--The staging process and the Y-maze experiment

Hello Everyone. My name is Sandra Ho and I am writing about my second and third week in the Evolution and Behavior Lab at Harvard University.

My second week was quite easy and simple. On the first two days I created more enriched and standard tubes for all types of flies for trials, which included: Canton.S and DGRP( include 45,105, 796 and 535). The flies in DGRP are not genetically identical as some of them exhibits higher variability and some of them exhibits lower variability. During the middle of the week, I noticed a lot of the flies in the trial tubes started to hatch. Ben suggested me to put them in the staging tubes for five days and then they would be available to test for behavior. I put 10 male and 10 female in each of the enriched tubes and 20 males or 20 females in each of the control tubes for staging. He also gave me a suggestion for my project: collect flies in outdoor by using traps. He thought it was a good idea to test them because those flies are wild types and the environment they used to live is a lot different from the flies in our lab, hence the results might be different. Therefore, I made five traps( put a paper funnel in the opening of the test tube which contains fly food) and put them behind a bush near my dorm.

The third week I finally got the chance to do the Y-maze experiment! Basically, the Y-maze experiment is to quantify left and right turning of the flies. An individual fly is placed in a Y-shaped maze on top of a light box, allowed to walk freely for two hours, and the X-Y position of the fly’s centroid  is tracked by a camera. The first thing I had to do was to make the flies asleep by using CO2 gas and use a brush to put them into a tray that consist of 120 individual Y-mazes. One fly in each maze and each maze is covered by a lid.This set up allows 120 flies to be run in parallel, and each of which makes hundreds of turns during the two-hour experiment Then I had to put the tray inside the light box and put all the information of the flies in the computer that is connected to the light box. And finally, when I clicked the button "Start Data Collection", the computer would then start collecting data!

This is the light box used for the Y-maze experiment.

This is the computer that is used for data collection.

And those are my trials and stocks of flies.

For the coming week, I will start analyze the data with Ben because there are soon enough flies that have been tested for behavior. I am really excited about it and I hope everyone is doing well in their lab too!

Week 5 to 7

Hi, this is Jacky and I am working in McAlpine Lab at Princeton. 

These three weeks have been very busy. Our projects have been moving to the first critical point, which is about measuring the quality of the thylakoids we make.

                The first test we perform is chlorophyll concentration measurement. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. Once the chlorophyll concentration is determined, the total chlorophyll yield should be determined by multiply the chlorophyll concentration in mg /mL times the volume (mL). Once the chlorophyll concentration and the total chlorophyll yield is known, the chlorophyll concentration should be adjusted by adding the appropriate amount of Washing Buffer or by centrifuging again the thylakoids and resuspending in the appropriate amount of Washing Buffer.

                By determining the concentration of chlorophyll we make, we can decide if the procedures we use is appropriate since there are a lot of different methods that we are able to choose from literature. It is always important in science research to try as much methods as possible.

                                          

                What’s more, we need to test the efficiency of the thylakoids we extract. In this case, we need to use a chemical method called Hill Reaction. As we learned in Biology class, during light reaction photosynthesis, electrons will end up in NADPH. In Hill reaction, we need to separate thylakoid with stroma so that NADP won’t be available. Instead, we will put DCPIP, a blue oxidant, into the solution. DCPIP is blue in its oxidized form, and becomes colorless when it is reduced during the Hill reaction. Thus, the rate at which electron transport occurs in the Hill reaction can be measured spectrophotometrically (at 620 nm) by following the change in absorbance of DCPIP as it accepts electrons from the electron transport chain. To perform the Hill reaction, a sample of a chloroplast suspension will be mixed with the Hill reaction buffer (containing DCPIP) and exposed to light for a series of 30 second intervals. After each exposure period, the absorbance of the DCPIP will be measured. The absorbance values can then be plotted versus time to determine the rate of DCPIP reduction as a measure of PET.

It turns out that the thylakoids we make are pretty efficient. We will keep on testing and trying for next couple days and shift our focus to other aspects. 

Problems and Solutions (Week 6-7)

Hey guys! This is Rhea at CHOP, working on Mitochondrial Disease research.

Because I discovered that the mutated worms we're working with are showing less signs of well..being mutated, Julian asked me to preform a lifespan experiment without using FUDR to figure out if that was the cause of the strange results. FUDR is a chemical used to stop cell growth and this prevents the worms being observed to reproduce and infest our plates with hundreds of worm larvae. Julian has been using FUDR for many years now and it has never shown affects on the lifespan of worms but there has been a paper published which reads that FUDR may affect worms. I set up a lifespan experiment without FUDR to check if it was the source of lifespan longevity and when I checked on it the following day it looked like this.

The big clumps on the plate are colonies of contamination (bacteria). 

All of my lifespan plates looked like this and so I had to discard them all (about 25 plates of 20 individually picked and placed worms).

Because now I won't have time to restart my lifespan, I've decided to take on a fluoresce project which I've watched Fred do a few times. He grows worms in plates with different dyes which illuminate the pharyngeal bulb and he takes pictures of them under a special microscope. I'll be analyzing pictures to see if the mutated gas-1 strain compares to previous results to make sure the gas-1 are truly gas-1... addressing what I had found with my lifespan. 

Computer Networking Lab weeks 6&7

Hi again, this is Sohan and I am working in a computer networking lab this summer at Columbia University.

These past 2 weeks have been more of the same. I have continued to run tests on the Orbit-lab, process the .pcap files using the Python scripts, and graph the results using the MATLAB scripts. Though it may seem monotonous and redundant, each time accessing the Orbit-lab presents its own adventure with its own unique challenges.

More recently, the nodes in the network are successfully uploading the requisite version of Ubuntu which solved one of my major problems before. However, other problems have occurred such as the nodes failing to communicate effectively with one another and sometimes the drivers not being installed properly (drivers are the device which helps the software communicate with the hardware and vice versa ex: the trackball on the older mouses interpreted the motion of the mouse and sent that information to the mouse driver which interpreted the signal and made the pointer on the screen move in the designated direction). In our case, the driver helps interpret the commands we are inputting and makes the hardware act accordingly ex. if we want the wlan card in the AP to release a signal of 18mbps, we input a command, the driver interprets the commands, and send a signal to the wlan device to release a signal of 18mbps. When the drivers are not installed properly, our commands may be ineffective and futile at points making the process of running the experiment very difficult. Nonetheless, there have been times where the Orbit-lab does in fact cooperate and work adequately and I have obtained data sets to analyze.

Also, over the past few weeks, Varun has had me debug/clean-up/develop the Python and MATLAB scripts. Some of the information previously had to be manually inputted such as the time/date and now, I have made them so that they can obtain the information directly from some of the files in our data sets. Similarly, since we use 2 Python scripts to process the .pcap files and one script is based off of the outputs of the previous script, I have made them more automated where the last script can retrieve the data from the text files outputted from the first script to make the process less complicated and help it run more smoothly.

Lastly, one of the members of the lab is leaving for Korea tomorrow. He will be starting graduate school in the fall at Michigan and will spend the rest of his summer at home in Korea. We had a nice little going away party for him this past week and at the same time welcomed a new member to our lab who will essentially take his place from China.

So far, work has been going well and hopefully, we have some more productive weeks ahead!

Chandran Laboratory Week 1: Dawn in Morningside Hights

So unlike my peers, who are mostly finishing up their lab work, I have only just begun my work at the Chandran Laboratory at Columbia University, working with a graduate student studying ammonia oxidizing bacteria and nitrous oxide and nitric oxide emissions.

 My new role as a commuter began when I was dropped off at the PATH station, gearing up for what I assumed to be a 2 hour commute to upper Manhattan. To my delight, everything went much faster than expected. I arrived and with only minimal trouble, found the correct building, and met my mentor, Medini, who is a first year graduate student and whose research I'm helping with. My PI, Dr. Chandran, was doing "field research" with another graduate student, and I would soon learn that he is often out of the office and very busy (although we did get a chance to meet to talk about the lab goals).

The first few days I passed the time observing Medini and her pure-culture batch reactor. Thus far all that she had been doing is observing the cell growth and troubleshooting reactor problems. The machinery is pretty cool, with the tub full of the media and cells connected via all these tubs to a fancy machine that regulates everything and shows all the things happening for you, except cell growth and product formation, which would be our job to track. Basically, her work involves Nitrosonomas eutropha, which is an ammonia oxidizing bacteria vital in nitrification, which is the change of ammonia into nitrate (via a few intermediate steps). Because the batch is a pure culture, meaning that no other bacteria are growing in it, this means that every piece of equipment and everything around it has to be extremely sterilized. Right off the bat, in order to retrieve a sample from the reactor, Medini had to use a Bunsen burner to switch out the tubing. Until my safety training, I could only observe, take notes, and hopefully absorb some skills.

Friday came quickly, and just in time for a new cycle of cell growth to begin. We had to make new media and autoclave many things and will soon be inoculating the reactor. While the cells grow in the reactor, we will be first observing their batch growth curve, which will involve lots of cell counting. Then we will be looking at product formation, in this case nitrite, using a spectrophotometer. Once the cell population stabilizes, then the real experimentation can begin.

 Fortunately, the heat wave is subsiding and I am particularly grateful seeing as I'd have to walk through the heat in my lab-appropriate long pants and cardigans. Today I met another high school student who is beginning a two year stint in the lab for INTEL and other lab research work, so it's good to see a familiar unsure face. The rest of the lab is graduate students and post-doc's from all over the world. According to Medini, it's one of the most diverse group of people around. From China to Brazil to New Jersey, the lab guarantees a lot of learning experience.

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.

On to the Next Phase-- Weeks 4-6 at the Murphy Lab

Hi everyone, I'm Richard and I'll be talking about my past 3 weeks spent at the Murphy Lab, which examines various aspects of the aging process in C. elegans.

Week 3 was a definite struggle.  I ran 3 STAMs with my egl-4 mutants and wild type worms, all of which produced results opposite from what I expected, and since the entire lab was in California at the International C. elegans meeting, I was pretty much on my own when trying to figure out what exactly went wrong.  Geneva hypothesized that the worms were too young, so I made sure to bleach the egl-4 mutants a few hours earlier than I had previously done, and luckily, I was able to confirm my expected result in another STAM.  The rest of the week was spent primarily on preparation for future experiments and analyzing my data.

Week 5 was, for the most part, uneventful.  Originally I had planned to do PCR and run a gel to confirm that my egl-4 crh-1 mating worked to produce double mutants, but since a previous PCR and gel showed that I had no double mutants, I decided it would be best to re-do the original cross. So, once again, the week was spent mostly preparing for the next phase of my project, examining EGL-4::GFP nuclear localization.

For the nuclear localization assay, I first starved my egl-4::GFP worms (these are not the same as egl-4 mutants; rather, they are wild type worms with the EGL-4 protein tagged with GFP so that fluorescence can be observed).  I waited an hour, as I usually do for my chemotaxis assays, and then put the worms on conditioning plates spotted with butanone.  After another hour, I transfered these worms onto a hold plate and prepared microscope slides as quickly as possible to ensure the worms didn't lose their food-odorant association, and then examined fluorescence under a microscope.  To do this, I first locate the head of the worm, at about 60x magnification, and then switch to the option that allows me to view the RFP tagged (red color) AWC neuron.  At this point, the entire neuron, except for the nucleus in the center, flashes a bright red.  I then switch to the option that allows me to view GFP (green color), and depending on whether the nucleus is a bright green or still dark, as observed under RFP, I can tell whether or not the EGL-4 protein has entered the nucleus. I repeated this procedure with naive (untrained) worms, and also with adapted worms (adaptation is when a long, repeated exposure to a certain odor actually diminishes the worms' response to that same odor).  Today I will be analyzing the images I took during these nuclear localization assays.

Overall, I've enjoyed my experience in lab.  However, my days are not always as busy as one would expect, and I've had a couple of setbacks with various aspects of my project, which have consequently resulted in less eventful days.  I guess failure is something every scientist has to deal with sooner or later.

Linksvayer Lab week 6- Gels and why I don't like them

Hi my name is Ben Wagner and I've been working in the Linksvayer Lab at Penn. We work on evolutionary biology and collective decision making in ants.

Although I still have 2 weeks left in the lab, I feel like I've already learned what will be my most important lesson from this summer. This lesson is simple: Science takes forever. I'm not saying this is a negative way (necessarily) but more of an important lesson all future exp-ers and scientists should know. I have had multiple hour long incubation and waiting periods, ant feeding periods that took up most of my day, and my project expand until it is 10 times the original size, with 20 times as much work.

But regardless, week 6 was an excellent week, although it started off very badly. The week was a week of PCR and Gel electrophoresis, which shouldn't be bad. PCR is easy, gels are fine, just a lot of waiting time, but that's okay! PCR appeared to be running correctly We had already run a number of gels, and the creation of these gels lead to spills, leaks, and a small TAE buffer explosion in the microwave. But that's okay, nothing serious and its all in the name of science! Probably half our samples had been tested in at least one marker in the gel (so 1/8th what the final sample amount will be). 
Then, A talk with an old lab Post-Doc, Luigi, resulted in my learning that all the PCR i had run so far, and tested on the gels, was completely wrong. It pretty much came down to my strands had been coming out at somewhere around 50 bp, which Riley and I thought was correct. Nope!   They should be around 250-300. So we decided to find the issue. We started single-plexing (one marker instead of multiple), fiddled with the marker amount, changed temperatures in the thermocycler. Then, Dr. Linksvayer gave us the advice to use use 4uL DNA, instead of the 1uL that multiple protocols told us to use. Now we have have successful results on the gel!!! The next step is to see if this will work with multiplexing, because otherwise this project will not be finished anytime soon.

With only 2 weeks left, I'm not so sure this project will be finished, especially because Dr. Linksvayer wants me to help with behavioral things in those weeks. And because we haven't actually submitted anything to sequencing yet, which normally takes about a month, and then after that statistical tests need to be run to make a photogenic tree.... So it won't be done by the time I am. But maybe data collection will be. We'll just have to see. Hope all my fellow exp-ers have had a good summer in their labs, I'll keep you guys posted.