Richard again, continuing on from my last post.
My last week was a week with more experiments than usual. On Monday, I did an STAM with the unc-73 mutant and wild type worms, and then tested chemotaxis up to two hours after training. I analyzed the images I took of the chemotaxis plates on Tuesday, and bleached my egl-4::GFP worms with the hope of doing one last adaptation assay on Friday. From what I observed, the unc-73 mutant did not make the food-odorant association as effectively as wild type, but it retained its memory better than wild type did, which is consistent with what Geneva found.
On Wednesday, I did the egl-4 and wild type STAM again, since there were various things that went wrong with my egl-4 strain in the past, and after analyzing my results, it seems that I have good data that confirms what is expected. I did the goa-1 and wild type STAM on Thursday, and like the unc-73 mutant, the goa-1 worms didn't learn as effectively, but retained a longer lasting association between food and butanone. Today I was hoping to do one last adaptation assay, but the worms that I had bleached were overgrown, and thus many had died, leaving only the young ones with very few eggs as the worms I had to bleach. As a result, the worms grew very poorly, and I had to spend much of the day analyzing my data from Wednesday.
Overall, reflecting on these past nine weeks, I felt I've learned a lot. Doing chemotaxis assays can get a bit routine, but it is nevertheless exciting to test new mutants and see what their learning and memory is compared to wild type. I was really hoping I could test the egl-4 crh-1 double mutant, since no one else has done that so far, but sometimes matings don't work out. Still, a lot of the STAMs I performed in the last few weeks were useful replicates for Geneva, and she is in the process of publishing her paper, which she said will most likely have me as one of the authors, since I produced some data that is of use. And that's really cool, because not many high schoolers can say that they've been published.
Anyway I'd just like to thank Dr. Murphy for giving me the opportunity to work in her lab, Geneva for mentoring me all while she is dealing with her pregnancy, Dr. Peretz for helping me throughout the year, and everyone else involved. Time to enjoy the last couple of hours I have left in lab.
Showing posts with label Richard Jin. Show all posts
Showing posts with label Richard Jin. Show all posts
Friday, August 9, 2013
Murphy Lab- Weeks 7-8
Hi, this is Richard, and I'm studying learning and memory at Princeton. I will break up the last three weeks I spent at the Murphy Lab into two posts, and this is the first of the two.
I spent the majority of my seventh week repeating the egl-4::GFP nuclear localization assays that I had started earlier. Since we don't really know what is supposed to happen in regards to the GFP-tagged EGL-4 protein entering the nucleus, I wasn't able to make too much sense of my results for my naive and trained worms. In some cases, the GFP localized into the nucleus of the AWC neuron, thus causing the nucleus to flash a bright green, and in other cases, it was mostly the surrounding cytoplasm that was lit up. Such ambiguity held true in both the naive and trained worms. We did however, know what to expect with my adaptation assay, so we tried to confirm this result in which for adapted worms, EGL-4 enters the nucleus and for mock adapted worms, it doesn't. However, while in some of the worms the EGL-4 protein had clearly localized into the nucleus, in other cases it seemed that it was present throughout the entire cell, not just the nucleus, as evidenced by the entire neuron being bright. Unfortunately, I didn't have enough time to repeat this again and get a definitive result.
I tried my egl-4 and crh-1 cross again, and got to the point where I had candidates who had a 1/16 chance of being double mutants, but I did not have the time to run a PCR and isolate the successful candidates that I could use for my double mutant chemotaxis assay. I did a PCR earlier, but none of the 30 candidates I picked out were identified as double mutants, and within a 9 week timeframe, it's pretty difficult to start from scratch and set up the same cross again. I was really looking forward to this, because, as of know, according to wormbase.org, pretty much the bible of C. elegans research, no one has tested egl-4 crh-1 double mutants. I could've been the first. But not anymore.
Anyway, I spent the rest of the two weeks on a couple of STAMs and an LTAM as well, to help with Geneva's project. The difference between an STAM and an LTAM (long term associative memory) is that rather than training the worms once (starve, food w/butanone, test), I must train them seven times in 30 minute intervals (starve, train, starve, train...). I didn't test chemotaxis, but Geneva used the worms I trained to observe fluorescence.
These two weeks were definitely more eventful than some I've had in the past.
I spent the majority of my seventh week repeating the egl-4::GFP nuclear localization assays that I had started earlier. Since we don't really know what is supposed to happen in regards to the GFP-tagged EGL-4 protein entering the nucleus, I wasn't able to make too much sense of my results for my naive and trained worms. In some cases, the GFP localized into the nucleus of the AWC neuron, thus causing the nucleus to flash a bright green, and in other cases, it was mostly the surrounding cytoplasm that was lit up. Such ambiguity held true in both the naive and trained worms. We did however, know what to expect with my adaptation assay, so we tried to confirm this result in which for adapted worms, EGL-4 enters the nucleus and for mock adapted worms, it doesn't. However, while in some of the worms the EGL-4 protein had clearly localized into the nucleus, in other cases it seemed that it was present throughout the entire cell, not just the nucleus, as evidenced by the entire neuron being bright. Unfortunately, I didn't have enough time to repeat this again and get a definitive result.
I tried my egl-4 and crh-1 cross again, and got to the point where I had candidates who had a 1/16 chance of being double mutants, but I did not have the time to run a PCR and isolate the successful candidates that I could use for my double mutant chemotaxis assay. I did a PCR earlier, but none of the 30 candidates I picked out were identified as double mutants, and within a 9 week timeframe, it's pretty difficult to start from scratch and set up the same cross again. I was really looking forward to this, because, as of know, according to wormbase.org, pretty much the bible of C. elegans research, no one has tested egl-4 crh-1 double mutants. I could've been the first. But not anymore.
Anyway, I spent the rest of the two weeks on a couple of STAMs and an LTAM as well, to help with Geneva's project. The difference between an STAM and an LTAM (long term associative memory) is that rather than training the worms once (starve, food w/butanone, test), I must train them seven times in 30 minute intervals (starve, train, starve, train...). I didn't test chemotaxis, but Geneva used the worms I trained to observe fluorescence.
These two weeks were definitely more eventful than some I've had in the past.
Monday, July 22, 2013
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.
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.
Monday, July 1, 2013
STAMs-- weeks 2&3 at the Murphy lab
Richard here, studying learning and memory in C. elegans in Dr. Murphy's lab at Princeton.
For the past two weeks, the primary focus of my research has been to perform short term associative memory training (STAMs) on wild type (N2) and egl-4(ky95) worms. I may have given a brief description of STAMs in an earlier post, but I'll go into more detail this time:
The experiment starts off with several plates of a strain of worms, which have been bleached so that the worms being tested are all about the same age. Using M9 buffer, I wash the worms off one of the plates to serve as my naive testing group-- I examine the worms' responsiveness to the chemical butanone without any prior conditioning which will enhance their response. Then, I wash the rest of the worms off the plates and into a 15 mL tube, where they starve for about an hour. When this hour has passed, I transfer the worms onto several conditioning plates, with food, and spot a small amount of butanone on each plate so that after an hour of conditioning, the worms will have a developed a strong association between food and butanone. To test this association, I prepare chemotaxis plates, which look like this:
The dots on the right and left are both spotted with sodium azide, which stops the worms from moving, and the right and left dots are spotted with ethanol and butanone, respectively. I put about 100-300 worms on the dot at the bottom, called the origin, and after an hour, I take a picture of the plate and use a digital sorting program to count the worms. This entire process is called a chemotaxis assay, and I need to perform one at various time points: right after conditioning (0 hr), 30 minutes after conditioning, 1 hr, 2 hr, 4 hr, and 6 hr, using three chemotaxis plates per time point per strain in order to ensure accuracy. The worms being tested at later time points are placed onto hold plates which contain food but no butanone. Factoring in the two hours it takes to starve and then train the worms, the entire experiment lasts 8 hours. However, there are large chunks of time in between, which gives me time to perform other preparations needed for future STAMs, and time to just relax.
My second week, I performed three STAMs with just the wild type strain, to get acquainted. My third week, I performed three STAMs with both the wild type strain and the egl-4 strain. It is known that the egl-4 strain retains its association between food and butanone for several hours, unlike wild type, which loses this association almost completely 2 hrs after conditioning, but during all three of the experiments I performed last week, the egl-4 strain actually displayed even worse chemotaxis towards butanone than did wild type, which is the exact opposite of what was expected. Two possible explanations immediately come to mind: 1) I am a genius who has just proven the scientific world wrong, or 2) I'm an idiot who, despite having performed the same exact experiment three times, still managed to completely screw up. Option number 1 sounds flattering, but is highly unlikely. Option number 2 seems more realistic, but while there are many things at which I am inept, I am certainly no idiot, never have been, and I am 150% sure that I performed these experiments with as impeccable timing as I could achieve. With this being said, at this point, I'm not sure what exactly went wrong, but now that my grad student, Geneva, along with the rest of the lab, is back from the International worm meeting at UCLA, hopefully we can figure something out.
In the meantime, I was able to cross my egl-4 mutants with my crh-1 mutants to produce heterozygotes, and then allow those to self fertilize to produce possible double mutants. In order to verify if any of the offspring chosen are indeed double mutants, I performed PCR and then ran a gel. However, if there are serious problems with my egl-4 strain, as evidenced by my extremely odd results from my egl-4 STAMs, then this pursuit may be in jeopardy. All I can do is hope for the best.
For the past two weeks, the primary focus of my research has been to perform short term associative memory training (STAMs) on wild type (N2) and egl-4(ky95) worms. I may have given a brief description of STAMs in an earlier post, but I'll go into more detail this time:
 |
| Chemotaxis assay of wild type right after conditioning-- all the worms are attracted to butanone (left) |
My second week, I performed three STAMs with just the wild type strain, to get acquainted. My third week, I performed three STAMs with both the wild type strain and the egl-4 strain. It is known that the egl-4 strain retains its association between food and butanone for several hours, unlike wild type, which loses this association almost completely 2 hrs after conditioning, but during all three of the experiments I performed last week, the egl-4 strain actually displayed even worse chemotaxis towards butanone than did wild type, which is the exact opposite of what was expected. Two possible explanations immediately come to mind: 1) I am a genius who has just proven the scientific world wrong, or 2) I'm an idiot who, despite having performed the same exact experiment three times, still managed to completely screw up. Option number 1 sounds flattering, but is highly unlikely. Option number 2 seems more realistic, but while there are many things at which I am inept, I am certainly no idiot, never have been, and I am 150% sure that I performed these experiments with as impeccable timing as I could achieve. With this being said, at this point, I'm not sure what exactly went wrong, but now that my grad student, Geneva, along with the rest of the lab, is back from the International worm meeting at UCLA, hopefully we can figure something out.
In the meantime, I was able to cross my egl-4 mutants with my crh-1 mutants to produce heterozygotes, and then allow those to self fertilize to produce possible double mutants. In order to verify if any of the offspring chosen are indeed double mutants, I performed PCR and then ran a gel. However, if there are serious problems with my egl-4 strain, as evidenced by my extremely odd results from my egl-4 STAMs, then this pursuit may be in jeopardy. All I can do is hope for the best.
Friday, June 14, 2013
Worms, Bombs, and Everything in Between
My first day was a bit of a surprise. After extensively studying and ultimately writing my review on the insulin-like growth factor 1 (IIS) pathway and its roles in aging in C.elegans (a nematode), I learned that the project which I would be undertaking had nothing to do with the pathway. But I'm actually glad that Dr. Murphy pushed me in a different direction. Before coming to the lab, I couldn't really decide which of the various components of the aging process, including reproductive aging, oxidative stress, and learning and memory, I wanted to focus on, so I simply said that I wanted to do something on general longevity regulation, which, in hindsight, was probably the least attractive of my options. I was assigned to work with a grad student, Geneva, who focused specifically on learning and memory. We came up with a project in which I would train wild type, egl-4 (which functions in the AWC neurons), and egl-4 crh-1 double mutant worms to develop an association between a specific odor and food, and then examine if such an association is still present after a certain amount of time. Unlike the IIS pathway, which is so well documented, the pathway in which EGL-4 acts is not, so it'll be really cool to study something which isn't as well understood. And since I'll be working for 9 weeks, I might get to make more double mutant strains to test.
So basically, the rest of that first day consisted of some basic training of techniques, i.e. how to bleach the worms to obtain the eggs (which sucks for the worms because their bodies get completely dissolved), seeding plates (putting bacteria on them); a 2 hr long lab meeting, during which I was hopelessly lost; and reading 2 articles.
Tuesday was weird, to say the least. Geneva, who is six months pregnant, needed to get some sort of test from a doctor, so she wasn't coming in until 12. I arrived at 8:30 as always, and got cracking on reading those last 2 articles I was supposed to. Some time between 10 and 11, I overheard two people in the lab I was in (not the Murphy lab because Geneva's office is in another lab) mention a bomb threat. I thought this was simply a drill, so I just sat at my desk and turned to Gunnar, a postdoc in Dr. Murphy's lab who, like Geneva, has his office outside the lab, asking what to do. He wasn't sure either, but a lady told us all to get out, so we did. It wasn't a chaotic scene, however. There was no sense of panic among everyone, and once we got outside we saw a mass of people walking towards the parking lots. Thank God Gunnar was there, because he was able to drive me home. Otherwise I would've been wandering around like an idiot.
Wednesday wasn't too eventful. I learned how to 'chunk' worms (cut out a piece of agar on a plate of worms and transfer it to another), but other than that, I didn't have much else to do in the lab, so I mainly sat in Geneva's office reading articles and learning more about C. elegans. In the afternoon Geneva and I went to another building, because she needed, for her project, to use a biosorter (a very fancy, intricate machine) to separate her worms which fluoresced under UV light (due to expression of GFP) from those which did not. There aren't too many low points when it comes to research, but this has to count as one of them. We essentially sat there doing nothing for three hours just waiting for the machine to count the tens of thousands of worms she had in her samples. And by the time I left about three hours later, the process still wasn't over.
The next day I attended safety training and I learned how to pick up individual worms, by using an extremely thin 'spatula' to scoop the worms up while I observed them under the microscope (these things are only about 1 mm long). At first I couldn't pick up a single one as I didn't want to puncture the agar on which the worms grew, but after a few minutes something just clicked and I was able to pick them up with ease. The funny thing is, on Friday, after I picked up the worms, they wouldn't get off the spatula, so I tried to scrape them off, but when I did that I would get agar stuck onto the spatula, which made it even harder for the worm to get off. There were several instances in which, after approval from Geneva, and a bit of guilt, I stuck the spatula into a gas flame and burned the worm to a crisp. Yes, I feel terrible about doing this, but I really had no other choice.
The rest of Friday was spent preparing for my first STAM (short term associative memory training) on Monday with wild type worms. I needed to bleach my worms (dissolve their bodies; keep the eggs to make sure all the worms I use are the same age), and seed my plates with E. coli. I also set up a mating of an egl-4 hermaphrodite and a crh-1 male so that I get heterozygotes in the next generation, from which I can ultimately select double homozygous recessive mutants.
So far I've enjoyed my time in the Murphy Lab. Next week will be busy, as I need to run three STAMs and do the necessary preparation for each (bleach worms, seed plates, chunk plates), but I'm looking forward to it. If there's one thing I'd say I don't like about the lab, it's that I have to work extensively with E. coli, and that stuff just smells terrible.
So basically, the rest of that first day consisted of some basic training of techniques, i.e. how to bleach the worms to obtain the eggs (which sucks for the worms because their bodies get completely dissolved), seeding plates (putting bacteria on them); a 2 hr long lab meeting, during which I was hopelessly lost; and reading 2 articles.
Tuesday was weird, to say the least. Geneva, who is six months pregnant, needed to get some sort of test from a doctor, so she wasn't coming in until 12. I arrived at 8:30 as always, and got cracking on reading those last 2 articles I was supposed to. Some time between 10 and 11, I overheard two people in the lab I was in (not the Murphy lab because Geneva's office is in another lab) mention a bomb threat. I thought this was simply a drill, so I just sat at my desk and turned to Gunnar, a postdoc in Dr. Murphy's lab who, like Geneva, has his office outside the lab, asking what to do. He wasn't sure either, but a lady told us all to get out, so we did. It wasn't a chaotic scene, however. There was no sense of panic among everyone, and once we got outside we saw a mass of people walking towards the parking lots. Thank God Gunnar was there, because he was able to drive me home. Otherwise I would've been wandering around like an idiot.
Wednesday wasn't too eventful. I learned how to 'chunk' worms (cut out a piece of agar on a plate of worms and transfer it to another), but other than that, I didn't have much else to do in the lab, so I mainly sat in Geneva's office reading articles and learning more about C. elegans. In the afternoon Geneva and I went to another building, because she needed, for her project, to use a biosorter (a very fancy, intricate machine) to separate her worms which fluoresced under UV light (due to expression of GFP) from those which did not. There aren't too many low points when it comes to research, but this has to count as one of them. We essentially sat there doing nothing for three hours just waiting for the machine to count the tens of thousands of worms she had in her samples. And by the time I left about three hours later, the process still wasn't over.
The next day I attended safety training and I learned how to pick up individual worms, by using an extremely thin 'spatula' to scoop the worms up while I observed them under the microscope (these things are only about 1 mm long). At first I couldn't pick up a single one as I didn't want to puncture the agar on which the worms grew, but after a few minutes something just clicked and I was able to pick them up with ease. The funny thing is, on Friday, after I picked up the worms, they wouldn't get off the spatula, so I tried to scrape them off, but when I did that I would get agar stuck onto the spatula, which made it even harder for the worm to get off. There were several instances in which, after approval from Geneva, and a bit of guilt, I stuck the spatula into a gas flame and burned the worm to a crisp. Yes, I feel terrible about doing this, but I really had no other choice.
The rest of Friday was spent preparing for my first STAM (short term associative memory training) on Monday with wild type worms. I needed to bleach my worms (dissolve their bodies; keep the eggs to make sure all the worms I use are the same age), and seed my plates with E. coli. I also set up a mating of an egl-4 hermaphrodite and a crh-1 male so that I get heterozygotes in the next generation, from which I can ultimately select double homozygous recessive mutants.
So far I've enjoyed my time in the Murphy Lab. Next week will be busy, as I need to run three STAMs and do the necessary preparation for each (bleach worms, seed plates, chunk plates), but I'm looking forward to it. If there's one thing I'd say I don't like about the lab, it's that I have to work extensively with E. coli, and that stuff just smells terrible.
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