In one of my last meetings with my PI, he told me that he hoped I learned something during my time in his lab. In the last six months, I have honestly learned so much.
This past summer was a crash course in cell and molecular biology for me. The graduate student I worked with explained so much to me that I learned in courses just this fall semester, and helped me understand these topics greatly. The lab technician taught me so many new lab techniques that I had never done before, and am now mostly comfortable doing on my own. The post doc I worked with guided my project, and frequently helped me learn how to troubleshoot issues.
I still feel as though I have found a wonderful community of scientists and researchers, and will always be welcome to return. I have already been invited to a potluck and a holiday party later this December, and to continue research in that lab if I ever have the time again.
I wish I could continue in the spring, but I will have too much going on and want to be able to dedicate the proper amount of time to my lab work there, which I do not believe I could do with my upcoming schedule.
I have learned so much about myself as a researcher during my six months at the Padiath lab, and I will be forever grateful for my time there.
Liquid Nitrogen, the Leidenfrost Effect, and Food?
Liquid nitrogen is extremely cold and allows the long term storage of cells when they are not being grown or used in an experiment. Liquid nitrogen seems very dangerous and should be treated with caution, but can be used safely in and outside of the lab. Wait, did I just say outside of the lab? You read correctly, some people use liquid nitrogen to do cool tricks - do not try these at home, but you can watch some examples below.
Someone even did the ALS Ice Bucket Challenge with liquid nitrogen instead, which had to have been so incredibly cold.
Others use liquid nitrogen for molecular gastronomy, which is essentially using principles of chemistry and physics to make interesting food and drinks. A very common dish is ice cream that is flash frozen by liquid nitrogen. Once the ingredients are mixed, liquid nitrogen is applied and the ice cream is ready in under a minute.
Liquid nitrogen has even made it to the bar, as a visual appealing addition to standard drinks.
While liquid nitrogen can be safe even when included in mixed alcoholic drinks, the liquid nitrogen must be completely evaporated off of the drink - just as the fire must be gone before something that has undergone flambé is consumed. If not, there can be immediate and severe effects, such as the degradation of the stomach.
Someone even did the ALS Ice Bucket Challenge with liquid nitrogen instead, which had to have been so incredibly cold.
Others use liquid nitrogen for molecular gastronomy, which is essentially using principles of chemistry and physics to make interesting food and drinks. A very common dish is ice cream that is flash frozen by liquid nitrogen. Once the ingredients are mixed, liquid nitrogen is applied and the ice cream is ready in under a minute.
Liquid nitrogen has even made it to the bar, as a visual appealing addition to standard drinks.
While liquid nitrogen can be safe even when included in mixed alcoholic drinks, the liquid nitrogen must be completely evaporated off of the drink - just as the fire must be gone before something that has undergone flambé is consumed. If not, there can be immediate and severe effects, such as the degradation of the stomach.
How can liquid nitrogen be used safely though, if it can also cause severe burns? Liquid nitrogen's boiling point is −195.8 °C, whereas room temperature is right around 20 °C, which is over a 200 degree difference. Because of this large difference, liquid nitrogen evaporates at a very high rate when exposed to room temperature, and will form droplets surrounded by its own vapor. This process is known as the Leidenfrost Effect.
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| Figure 1. An example of what evaporating liquid nitrogen would look like as it hits a room temperature surface (Walker, n.d.). |
References:
BBC News. 2015. Liquid nitrogen cocktail: Lancaster's Oscar's Wine Bar fined £100k. <http://www.bbc.com/news/uk-england-lancashire-34277429>.
Darryl's Wood Fire Grill. 2012. Liquid Nitrogen Mixed Drinks! <https://www.youtube.com/watch?v=T4GfdM9cKU8>.
Grant Thompson - "The King of Random." 2016. What does Liquid Nitrogen do to Your Face? <https://www.youtube.com/watch?v=l1XRspReAvI>.
Helmenstine, A. M. 2016. Liquid Nitrogen Facts.<http://chemistry.about.com/od/moleculescompounds/a/liquidnitrogen.htm>.
Helmenstine, A. M. 2016. Room Temperature Definition. <http://chemistry.about.com/od/chemistryglossary/g/Room-Temperature-Definition.htm>.
ImaginationStationOH. 2014. Liquid nitrogen ice cream. <https://www.youtube.com/watch?v=dNmMW4E54lI>.
MolecularRecipes.com. n.d. What is Molecular Gastronomy? <http://www.molecularrecipes.com/molecular-gastronomy/>.
NurdRage. 2014. ALS Liquid Nitrogen "Ice Bucket Challenge" (Ft. Muhammad Qureshi). <https://www.youtube.com/watch?v=Xj-prpHfyEY>.
NurdRage. 2010. Hand vs. Liquid Nitrogen - Revisited. <https://www.youtube.com/watch?v=wV7g8L633Sg&t=14s>.
Walker, J. n.d. Boiling and the Leidenfrost Effect. Cleveland State University. [PDF].
BBC News. 2015. Liquid nitrogen cocktail: Lancaster's Oscar's Wine Bar fined £100k. <http://www.bbc.com/news/uk-england-lancashire-34277429>.
Darryl's Wood Fire Grill. 2012. Liquid Nitrogen Mixed Drinks! <https://www.youtube.com/watch?v=T4GfdM9cKU8>.
Grant Thompson - "The King of Random." 2016. What does Liquid Nitrogen do to Your Face? <https://www.youtube.com/watch?v=l1XRspReAvI>.
Helmenstine, A. M. 2016. Liquid Nitrogen Facts.<http://chemistry.about.com/od/moleculescompounds/a/liquidnitrogen.htm>.
Helmenstine, A. M. 2016. Room Temperature Definition. <http://chemistry.about.com/od/chemistryglossary/g/Room-Temperature-Definition.htm>.
ImaginationStationOH. 2014. Liquid nitrogen ice cream. <https://www.youtube.com/watch?v=dNmMW4E54lI>.
MolecularRecipes.com. n.d. What is Molecular Gastronomy? <http://www.molecularrecipes.com/molecular-gastronomy/>.
NurdRage. 2014. ALS Liquid Nitrogen "Ice Bucket Challenge" (Ft. Muhammad Qureshi). <https://www.youtube.com/watch?v=Xj-prpHfyEY>.
NurdRage. 2010. Hand vs. Liquid Nitrogen - Revisited. <https://www.youtube.com/watch?v=wV7g8L633Sg&t=14s>.
Walker, J. n.d. Boiling and the Leidenfrost Effect. Cleveland State University. [PDF].
Cells Do Not Care About You, But You Should Care About Them
I do not yet know what it is like to work in other types of labs outside of cell biology and genetics, but I can say that working in a lab primarily with cells, you have to be willing to work weird hours. It is possible for me to schedule my time as I please, but cells will not wait for you. They do not care if you what plan you may have already made if they need to be split or cared for a specific day, they do.
Some cells will stop growing altogether once they have become too confluent, whereas others will grow on top of each other infinitely.
Some cells will stop growing altogether once they have become too confluent, whereas others will grow on top of each other infinitely.
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| Figure 1. Cells are pretty cell-fish, wouldn't you say? (Luong, 2014). |
If cells did have feelings, they probably would put you into their own Burn Book, like Gretchen Weiners from the movie Mean Girls. They'd be bullies. However, cells are not people, they do not have feelings or emotions, but they do make up every living thing in the world around us. Now, I don't want it to seem like I hate cells, because it's really just the opposite. Rather, I am willing to deal with the difficulties that come with cells because of how amazing they are, and how useful they are in research. Cells can allow me to find new treatments for diseases and disorders, which is simply remarkable.
References
Luong, C. 2014. Cell-fie. [Image]. <http://christineluong.tumblr.com/post/79189174454/apologies-for-inaccuracies-i-tried#notes>.
References
Luong, C. 2014. Cell-fie. [Image]. <http://christineluong.tumblr.com/post/79189174454/apologies-for-inaccuracies-i-tried#notes>.
Research Is Time Consuming
Continuing research during the semester is entirely possible, but it does take a lot of time and focus. If you want to conduct research while you are in classes, you have to be dedicated. Sometimes you may have to go in and conduct research even though you have a huge test coming up the next day, or you may have to miss an event that you really wanted to go to.
The amount of time you will have to spend on research depends on many factors:
1. Research Location. Travel is often necessary to be able to conduct the research that you want to, and it is important to consider the time it will take you to get to your research site into the time you will have to dedicate to your work. If you are conducting bioinformatics or related research that can be completed primarily or entirely on a laptop, you may have to travel very little, perhaps only for meetings. I personally travel to a different campus from where all of my classes and other events occur to conduct research.
2. Project Type. If you are working on your own independent project, then you will need to be at your research site quite frequently. If you are working as part of a team, you may need to be there at more specific times, but you will have more freedom in how much time you have to be at the your research site since your team can split up the work. However, you will spend more time coordinating with other people to make sure everything is getting done. I am lucky enough to be working with an undergraduate student who goes to school at the campus I travel to, so she is able to stop in for short tasks much easier than I can. We communicate via text and just update each other when necessary, which is proving to be very effective.
3. Requirements. Research can often count for credit, or function as a job, or even both depending on your situation. If you are receiving credit and/or a stipend for your research work, you will likely have a total amount of hours you must reach either overall or per week. It will likely not be difficult for you to reach your hour requirements, but it is important to keep this value in mind. I am receiving one credit for my time, so I need to reach 40 hours total over 14 weeks. I think I have reached this amount of hours already, only halfway through my semester.
At one point one of my professors at my home institution told me that there was no way you could do less than ten hours a week of research and actually get anything done, and that completely is true. A lot of research is waiting for procedures to complete, which simply takes time. The good news is that when you are waiting and have no other lab tasks to complete, you have a wonderful chance to get some homework or studying done for your classes, or catch up on other obligations if possible. Finding connections between your research and your coursework can make the time seem less overwhelming as well.
The amount of time you will have to spend on research depends on many factors:
1. Research Location. Travel is often necessary to be able to conduct the research that you want to, and it is important to consider the time it will take you to get to your research site into the time you will have to dedicate to your work. If you are conducting bioinformatics or related research that can be completed primarily or entirely on a laptop, you may have to travel very little, perhaps only for meetings. I personally travel to a different campus from where all of my classes and other events occur to conduct research.
 |
| Figure 1. What I imagine some of my friends think I am off to when I say I have to go to lab (Encyclopedia SpongeBobia). |
2. Project Type. If you are working on your own independent project, then you will need to be at your research site quite frequently. If you are working as part of a team, you may need to be there at more specific times, but you will have more freedom in how much time you have to be at the your research site since your team can split up the work. However, you will spend more time coordinating with other people to make sure everything is getting done. I am lucky enough to be working with an undergraduate student who goes to school at the campus I travel to, so she is able to stop in for short tasks much easier than I can. We communicate via text and just update each other when necessary, which is proving to be very effective.
3. Requirements. Research can often count for credit, or function as a job, or even both depending on your situation. If you are receiving credit and/or a stipend for your research work, you will likely have a total amount of hours you must reach either overall or per week. It will likely not be difficult for you to reach your hour requirements, but it is important to keep this value in mind. I am receiving one credit for my time, so I need to reach 40 hours total over 14 weeks. I think I have reached this amount of hours already, only halfway through my semester.
 |
| Figure 2. Most research conducted by undergraduate students during the semester will provide the chance to receive a stipend and/or college credit (Collegebound Network). |
At one point one of my professors at my home institution told me that there was no way you could do less than ten hours a week of research and actually get anything done, and that completely is true. A lot of research is waiting for procedures to complete, which simply takes time. The good news is that when you are waiting and have no other lab tasks to complete, you have a wonderful chance to get some homework or studying done for your classes, or catch up on other obligations if possible. Finding connections between your research and your coursework can make the time seem less overwhelming as well.
References
Collegebound Network. College Debt Surpasses Credit Card Debt. 2010. [Image]. <http://www.collegebound.net/blog/college-debt-surpasses-credit-card-debt/>.
Collegebound Network. College Debt Surpasses Credit Card Debt. 2010. [Image]. <http://www.collegebound.net/blog/college-debt-surpasses-credit-card-debt/>.
Encyclopedia SpongeBobia. Top Secret Laboratory. n.d. [Image]. <http://spongebob.wikia.com/wiki/Top_Secret_Laboratory>.
Back to the Lab
I'm back! I started to consider a path in research about a year ago but I could never have imagined where I am today already. I am incredibly excited to be contributing to research in the Padiath lab again and continuing my project after all I was able to accomplish this past summer. I will be sharing more of my experiences here throughout my continued research.
One of the major things I realized now that I have started classes again is how much theory I simply didn't know and was learning from scratch during this summer. I ended up in a Human Genetics lab as a chemistry major who had taken only introductory biology, yet my day-to-day activities were in molecular biology surrounding topics of genetics and neuroscience. I am so happy I received the lab placement I did - it was my top choice and my work was so interesting - but wow, I was missing so much background. I've found over time that being thrown into research situations that cover topics I have not previously learned about in detail actually help me retain the new information very well, since I everything I am learning is immediately applicable. Situations like that can be a whirlwind but so rewarding. However, I wonder if my time in the lab would have been different had I only needed to remember concepts rather than learn them entirely.
Since I am continuing my project from the summer and am no longer part of the fellowship program, I am working directly with the other undergraduate student and we are able to coordinate our time around our classes. It can be hectic for us, but it is nice to have someone else at my level - she is even in the same grade as me - to share the work with. The new lab technician has also started working in the lab, so my undergraduate peer and I have been officially moved to the room where the thermal cyclers are rather than our space on the lab bench in the open floor area. I can't really complain considering that I have to use one of the thermal cyclers during the Western Blot procedure to denature the protein by heat.
I have started working with cells again, so coming in on weekends is still necessary. However, each day that I am coming in, I only have to stay for about an hour or two, which is nice. I am so grateful to be able to continue lab work through this semester, even though I am in the midst of classes and other responsibilities. Since I am not at the lab nearly as much, we have only gotten on set of results so far, but thankfully they are promising. However, we were conducting the experiment to try to figure out why we had weird results during another related experiment, so I think the initial experiment will now need to be redone. We are now starting a new but very similar project as to what I had started in the summer, in which we are going through the same experimental process for another drug to see which is a more viable option to treat Autosomal Dominant Leukodystrophy, using cell cultures.
I have already presented my summer research project once through the Center for Neuroscience at Pitt as part of my fellowship, and I will soon be presenting again - with less detail to protect the secrecy of our work - to students and faculty at my home institution, Chatham University, as part of a lecture series focused on work that students conducted over the summer. I feel very lucky to be able to talk at my home institution in such a public way about my summer research experience, and hopefully I might even inspire some students at my university to pursue an internship or a research experience this upcoming summer, or even sooner. I submitted a picture from my internship for the event flyer - the picture is one of my favorites that the lab technician so nicely took of me during my summer experience.
I believe there will be about six or seven other students presenting their work from this past summer, including current sophomores, juniors, and seniors. Though I conducted research during the summer after my first year, it was in a very relaxed manner. I am fairly certain that the sophomore students presenting completed internships or working on research over this past summer, and I am so impressed that they were already so driven to dedicate their summers to science so early in their undergraduate career. I cannot wait to hear about my peers' summers, and probably will gush about their work further in a future blog post after the presentations are complete.
References:
ThermoFisher Scientific. n.d. Veriti 96-Well Thermal Cycler. <https://www.thermofisher.com/order/catalog/product/4375786>.
One of the major things I realized now that I have started classes again is how much theory I simply didn't know and was learning from scratch during this summer. I ended up in a Human Genetics lab as a chemistry major who had taken only introductory biology, yet my day-to-day activities were in molecular biology surrounding topics of genetics and neuroscience. I am so happy I received the lab placement I did - it was my top choice and my work was so interesting - but wow, I was missing so much background. I've found over time that being thrown into research situations that cover topics I have not previously learned about in detail actually help me retain the new information very well, since I everything I am learning is immediately applicable. Situations like that can be a whirlwind but so rewarding. However, I wonder if my time in the lab would have been different had I only needed to remember concepts rather than learn them entirely.
Since I am continuing my project from the summer and am no longer part of the fellowship program, I am working directly with the other undergraduate student and we are able to coordinate our time around our classes. It can be hectic for us, but it is nice to have someone else at my level - she is even in the same grade as me - to share the work with. The new lab technician has also started working in the lab, so my undergraduate peer and I have been officially moved to the room where the thermal cyclers are rather than our space on the lab bench in the open floor area. I can't really complain considering that I have to use one of the thermal cyclers during the Western Blot procedure to denature the protein by heat.
 |
| Figure 1. My new best friend, Veriti. When I go to lab now, I spend most of my time with this machine, even when I am not using it (ThermoFisher). |
I have started working with cells again, so coming in on weekends is still necessary. However, each day that I am coming in, I only have to stay for about an hour or two, which is nice. I am so grateful to be able to continue lab work through this semester, even though I am in the midst of classes and other responsibilities. Since I am not at the lab nearly as much, we have only gotten on set of results so far, but thankfully they are promising. However, we were conducting the experiment to try to figure out why we had weird results during another related experiment, so I think the initial experiment will now need to be redone. We are now starting a new but very similar project as to what I had started in the summer, in which we are going through the same experimental process for another drug to see which is a more viable option to treat Autosomal Dominant Leukodystrophy, using cell cultures.
I have already presented my summer research project once through the Center for Neuroscience at Pitt as part of my fellowship, and I will soon be presenting again - with less detail to protect the secrecy of our work - to students and faculty at my home institution, Chatham University, as part of a lecture series focused on work that students conducted over the summer. I feel very lucky to be able to talk at my home institution in such a public way about my summer research experience, and hopefully I might even inspire some students at my university to pursue an internship or a research experience this upcoming summer, or even sooner. I submitted a picture from my internship for the event flyer - the picture is one of my favorites that the lab technician so nicely took of me during my summer experience.
 |
| Figure 2. The protective gear I had to wear every time I went to work with the mice. This picture is now around my university's science building on the event flyer, and I just wonder what people think is in the container I am holding. |
I believe there will be about six or seven other students presenting their work from this past summer, including current sophomores, juniors, and seniors. Though I conducted research during the summer after my first year, it was in a very relaxed manner. I am fairly certain that the sophomore students presenting completed internships or working on research over this past summer, and I am so impressed that they were already so driven to dedicate their summers to science so early in their undergraduate career. I cannot wait to hear about my peers' summers, and probably will gush about their work further in a future blog post after the presentations are complete.
References:
ThermoFisher Scientific. n.d. Veriti 96-Well Thermal Cycler. <https://www.thermofisher.com/order/catalog/product/4375786>.
Week Ten
I have completed my research program! It has been an amazing experience, and I am so grateful I received this opportunity. This summer has allowed me to see that I do enjoy laboratory research, and that I have an aptitude for it as well.
I have been invited to continue researching in the Padaith lab in the fall, and I am quite excited. My main goal in scientific research is to find treatments for mood and neurodegenerative disorders and diseases that severely affect quality of life. Right now, I am studying a rare neurodegenerative disorder - Autosomal Dominant Leukodystrophy - and if I could help find a treatment for it, that would absolutely incredible. I think it is remarkable the impact that medical research can have on people's lives.
During the beginning of the week, I was still analyzing samples and collecting data for my project. Once I was done with that I was primarily working on my presentation and one last experiment for my project that would not be done in time for my presentation, but is important for the project itself. There will always be new information or data, so at some point you have to find a cut off point and keep that information for the next presentation, paper, or grant. One of the hardest parts of assembling my presentation was finding a way to include all of the information that I wanted and needed to within a ten minute window, which was the time allotted for each presentation.
The day before I was going to be presenting, I went to practice in the room where it would happen. I got to meet the newest assistant for the department my fellowship is through, and we talked for a bit. When I told her I went to Chatham, she recognized it immediately because it is her partner's alma mater. Such a small world! The assistant herself is also a poet, as am I in the time outside of my scientific work, and it was great to be able to meet a fellow artist when I didn't expect to at all.
It was very interesting to hear about the other fellows' projects through their presentations. I have learned so much about neuroscience and what different research areas are encompassed in the field. Not only did the projects of the fellows cover many different scientific disciplines, but we represented majors including chemistry, biochemistry, cell biology, and psychology. Many of the fellows were conducting trials of drugs on cells like I was, some were working primarily with mouse and rat models to look at the impact of brain damage or diseases, and one fellow was working with human subjects on computerized tests of cognitive ability.
The Padiath lab members as well as other people on my floor also took me out for food a few times to celebrate the end of my program, which I appreciated. I am so happy that I have found such wonderful and caring people. I was also able to spend time with the other fellows in my program, and it was nice to hear about their backgrounds and future plans in a casual setting, outside of their presentation. Other than realizing that I enjoy lab work, I like the community I have seen in scientific research over these past ten weeks. I get to meet so many brilliant and kind people from so many different backgrounds. I have also been able to meet people from so many different countries just on my lab floor, and it shows me that science has the ability to connect us all.
I have been invited to continue researching in the Padaith lab in the fall, and I am quite excited. My main goal in scientific research is to find treatments for mood and neurodegenerative disorders and diseases that severely affect quality of life. Right now, I am studying a rare neurodegenerative disorder - Autosomal Dominant Leukodystrophy - and if I could help find a treatment for it, that would absolutely incredible. I think it is remarkable the impact that medical research can have on people's lives.
During the beginning of the week, I was still analyzing samples and collecting data for my project. Once I was done with that I was primarily working on my presentation and one last experiment for my project that would not be done in time for my presentation, but is important for the project itself. There will always be new information or data, so at some point you have to find a cut off point and keep that information for the next presentation, paper, or grant. One of the hardest parts of assembling my presentation was finding a way to include all of the information that I wanted and needed to within a ten minute window, which was the time allotted for each presentation.
 |
| Figure 1. Presentation Day. |
The day before I was going to be presenting, I went to practice in the room where it would happen. I got to meet the newest assistant for the department my fellowship is through, and we talked for a bit. When I told her I went to Chatham, she recognized it immediately because it is her partner's alma mater. Such a small world! The assistant herself is also a poet, as am I in the time outside of my scientific work, and it was great to be able to meet a fellow artist when I didn't expect to at all.
It was very interesting to hear about the other fellows' projects through their presentations. I have learned so much about neuroscience and what different research areas are encompassed in the field. Not only did the projects of the fellows cover many different scientific disciplines, but we represented majors including chemistry, biochemistry, cell biology, and psychology. Many of the fellows were conducting trials of drugs on cells like I was, some were working primarily with mouse and rat models to look at the impact of brain damage or diseases, and one fellow was working with human subjects on computerized tests of cognitive ability.
The Padiath lab members as well as other people on my floor also took me out for food a few times to celebrate the end of my program, which I appreciated. I am so happy that I have found such wonderful and caring people. I was also able to spend time with the other fellows in my program, and it was nice to hear about their backgrounds and future plans in a casual setting, outside of their presentation. Other than realizing that I enjoy lab work, I like the community I have seen in scientific research over these past ten weeks. I get to meet so many brilliant and kind people from so many different backgrounds. I have also been able to meet people from so many different countries just on my lab floor, and it shows me that science has the ability to connect us all.
Week Nine
I started my final Western Blot on Friday and let it run while I was at my lunch lecture. However, when I came back all of the members of the Padiath lab seemed very concerned with the progress of my gel - the proteins had stopped moving, even the ladder was no longer moving. The lab tech asked me if I had heated the protein samples before loading them, and I hadn't - it was something I forgot to write in the notes I was following so it completely slipped my mind. Without heating the proteins, they do not become denatured and even if they had continued to run down the gel during the electrophoresis stage, the locations they ended up on the gel would have been inaccurate because the size of a protein folded into tertiary or quaternary structure is quite different from the length of its unfolded primary structure as an amino acid chain. The reason the proteins were not moving was because our apparatus and power sources have been not been working as they should, and sometimes stop the electrophoresis from progressing. Since I already missed an important step, it was actually helpful that the equipment malfunctioned because otherwise I may have gone through the rest of the Western Blot and only realized I had missed a step once I was analyzing the results.
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| Figure 1. Simplified drawings comparing folded and denatured proteins (RSC). |
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| Figure 2. Outline of the plasmid cloning process (Addgene). |
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| Figure 3. Dopamine, a chemical crucial to proper function and response of the reward system of the brain. |
I had to write an abstract for my project this week, and will give a presentation on my work next week. Writing the abstract allowed me to collect my thoughts and gain a better understanding of the relevance of my project to the disorder I am studying. My abstract will serve as a summary of my time in this lab and all I have achieved as I have been here, while my presentation will allow me to share my work with the other fellows and the directors of my program, as well as any other guests that attend, including my mentor and possibly one of the Padiath lab members. I will be collecting data for my presentation right up until it is due, as well as some data for my project that I will only be able to collect after my presentation is due, but is important for the future of the project.
Wednesday was a fun day because we had a birthday party for one of the women who works on my floor as a lab tech and is also a graduate student in our department. Some of the people on our floor bought her cakes from Oakmont Bakery that we were all encouraged to eat, which was a nice break during the day. She is incredibly friendly and talkative, and I am happy to have been able to make a connection with her and other people who work on our floor, even though they do not all work in the Padiath lab.
References:
Addgene. n.d. Plasmid Cloning by PCR. <https://www.addgene.org/plasmid-protocols/pcr-cloning/>.
[RSC]. n.d. Enzymes. <http://www.rsc.org/learn-chemistry/resources/chemistry-in-your-cupboard/vanish/8>.
Week Eight
Thankfully, I figured out early in the week what had been killing the cells I have been working with. I had run out of the plates I had been using for the cells, and assumed that a slightly different type of plate would be fine - they were same diameter as what I had been using and were kept in the same drawer. However, I did not realize that the new plates I decided to use were too different, and did not have any substance on them that allowed the cells to attach. It is likely that the cells were not even dying, but since they were not attaching it seemed as though they were dying, because usually that's how we determine that cells have completely died. Regardless, the grad student corrected my mistake and my cells have been growing fine since I switched back to the correct plates. When people tell you to not make any assumptions in science, listen. Even the smallest assumptions can cause large problems. In this case, I mostly lost time, so the possibility that I would have to come in over the weekend became a definite.
I started working with fibroblasts now, which are cells found in connective tissues. Our particular cells are not from an immortal cell line like the HEK 293s, so we have to avoid splitting them as they will stop growing properly after too many passages. It is exciting to have started my work with the fibroblasts since they are the next step in the determining how effective the treatment I am testing is. It is also quite interesting to work with a new cell type, since my work during this summer experience is the first time I have worked with cell cultures, and it is always fascinating to see cells under the microscope as they grow.
Last week we had attempted to replicate certain plasmids that we had through a transformation with bacteria. In a transformation, the bacteria used take in the plasmid DNA and as the bacteria replicates - which it does quickly - the plasmid DNA replicates as well. After the bacteria has replicated enough, the plasmids can be isolated again from the bacteria and used for other purposes. We performed the isolation this week and the DNA concentrations were what we expected, showing that we had successful replicated the plasmids. One of the major concerns in a transformation is that only the bacteria that has taken in the plasmid is what should be replicating, otherwise it is impossible to differentiate which bacteria actually has the plasmid in them or not. The plasmid has an antibiotic resistant gene in it that is expressed after a short time once it has entered a bacterium, so the addition of antibiotics to the media in which the bacteria will grow is sufficient to kill any unwanted bacteria.
On Monday of this week, once I had resolved the issue with my cells, I was ready to work and decided to start the Western Blot I needed to complete. In our lab, Western Blotting takes two days to complete once protein samples have been collected, with the first day being the longest. Even though I started the Western Blot at noon, it really was too late since the entire protocol for the first day - with many waiting periods - takes about seven hours, which I hadn't fully realized until I had started. The lab technician I work with was going to warn me, but honestly it wouldn't have mattered much if she had - I was determined to start the Western Blot and I didn't mind staying late that much, especially considering the protocol is not one where you actively must be involved the whole time. The extended time just meant that I would go home later that day, but have my results sooner and have a calmer week overall past that point. One of the hardest parts of working long hours when you haven't anticipated it before you left for the lab is not having enough food for the time you are there. However, later in the day the lab tech and graduate student were still there with me. the graduate student had a moment to run out and went to get snacks - I thought for himself - and came back with a dozen donuts, which he offered to the lab tech and I. It was a small gesture but he really didn't have to bring us back food, and it made any doubt about my decision to stay late go away.
References:
Addgene. n.d. Bacterial Transformation. <https://www.addgene.org/plasmid-protocols/bacterial-transformation/>.
CELLnTEC. n.d. Primary Human Dermal Fibroblasts. <http://cellntec.com/products/skin/fibroblast-cells/>.
Van Alest, K. 2005. Cellular mitosis. Photograph. <http://www.kevinvanaelst.com/photo10.html>.
I started working with fibroblasts now, which are cells found in connective tissues. Our particular cells are not from an immortal cell line like the HEK 293s, so we have to avoid splitting them as they will stop growing properly after too many passages. It is exciting to have started my work with the fibroblasts since they are the next step in the determining how effective the treatment I am testing is. It is also quite interesting to work with a new cell type, since my work during this summer experience is the first time I have worked with cell cultures, and it is always fascinating to see cells under the microscope as they grow.
 |
| Figure 1. Extremely confluent human dermal fibroblasts; their function in connective tissues apparent in their morphology and growth patterns (CELLnTEC). |
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| Figure 2. The transformation process for replicating plasmids (Addgene). |
 |
| Figure 3. Mitosis illustrated with donuts (Van Alest, 2005) |
Addgene. n.d. Bacterial Transformation. <https://www.addgene.org/plasmid-protocols/bacterial-transformation/>.
CELLnTEC. n.d. Primary Human Dermal Fibroblasts. <http://cellntec.com/products/skin/fibroblast-cells/>.
Van Alest, K. 2005. Cellular mitosis. Photograph. <http://www.kevinvanaelst.com/photo10.html>.
Week Seven
This week was quite busy and I was able to perform many new procedures, including real-time PCR and immunohistochemistry. Both processes were similar to one I have done before, standard PCR and Western Blotting, respectively. I have also become aware that, just like cells need care even when it's inconvenient, sometimes procedures will take you straight through when you wanted to eat lunch or late into the day and you have to figure out how to handle that. It usually it not as hard if you are busy - you can easily pass hours in what feels like minutes in a lab, especially if there are no clocks around you. However, working like that for hours straight - especially without eating much - can be tiring, so it is important to try to complete your most crucial tasks when you are most awake, in the morning or early afternoon, and to try to take breaks when you can. There are waiting periods in many procedures, and these are great chances to go get a drink of water or to eat a snack - or, if you're really tired, to go get some coffee. It is also good to keep in mind how long a certain procedure takes before you start it, so you know what you're getting into and if you are choosing a good time to start it with whatever else you have planned out for your day or week.
Real Time PCR is a method that is often used for analysis of RNA samples, since standard PCR does not really work with RNA itself, and doing the PCR analysis in real time instead of with a gel afterwards provides quantitative data that can be later manipulated with statistics - again, another use of Excel. Since the starting material is RNA, it must first be converted to cDNA through a reverse transcription process before it can be used for real time PCR. Real time PCR utilizes a machine that can monitor the polymerase chain reaction as it happens by noting when it sees the samples fluorece as the cDNA has progressed through another cycle. For our machine we must use 384 well plates, which have very small wells and can be a bit disorienting at first. Thankfully, our lab has a few multichannel pipettes which makes it so we can load multiple sample at once. Real time PCR seemed like a daunting task to me before I had tried it, but once I had I realized it was not as difficult as I thought it would be. The advice that the graduate student in my lab gave me was that the hardest part is simply remembering your place on the plate, and I feel like that is true for most procedures where you have to load samples, though it is a bit harder since there are so many more wells for this than ever would be for a standard PCR or a Western Blot.
Immunohistochemistry is one of those lab procedures that not only can provide important information about the morphology of cells, but is also quite fascinating and reminds me how science can allow us to look at parts of our world that we normally cannot. During many of the Friday lunch lectures that the mentors have been giving, they have been excitedly showing us pictures of cells that have been stained and imaged - even people who have their Ph.D.s already and have been looking at cells for years are still amazed by the beauty of these pictures. There are many different dyes that can be used for immunohistochemistry, and the the fluorescent dyes give some of the most interesting pictures. Two are shown in below: the DAPI fluorescent dye stains the nuclei of cells, whereas phalloidin 448 stains the actin and the cytoskeleton (ThermoFisher).
The protocol for immunohistochemistry was explained to me initially by the technician I work with in regards to its is similarity to Western Blotting, since it requires the use of a primary and secondary antibody. The overall procedure combines many different techniques, as first cells must be grown and treated appropriately, then cover slips have to be probed so they can imaged under a microscope that is attached to a computer that allows pictures to be taken and distributed.
I've again been having issues, this time with my cells. A few weeks ago, I had frozen down cells to store them and have been plating them for my project. The first time I plated them everything went fine, and I continued to grow them for a bit. One of the later times when I was splitting my cells, I believe that I did not do it aggressively enough and when I checked the dishes later under the microscope, I looked around the entire plate and could find no cells. I had been leaving plates in the incubator with only media in them as I must have left the cells in the plate I discarded when trying to split them. However, I was not incredibly concerned because I still had more aliquots of my frozen down cells, and I simply plated one of those. When I checked those cells, they were absolutely dead. I tried plating more cells, and they were also dead. Next week I will be attempting to plate cells again, using aliquots that someone else had froze down in case an issue occurred when I froze the cells down. Something could also be going wrong as I am plating the cells, but overall I just hope that the cells will adhere to the bottom of the plate and grow as they should, as I need them to complete the next tasks for my project.
References:
Bruce, D. 2013. Cell culture and stem cell techniques. <http://www.laney.edu/wp/doug_bruce/cell_culture_stem_cell_training/>.
E&K Scientific. 384 Well Roche 480 Plate, White, Barcoded. <http://www.eandkscientific.com/384-Well-PCR-Plate-White-for-Roche-Light-Cycler-Barcoded.html>.
Labnet. BioPette Plus Autoclavable Multichannel Pipettes. <http://northamerica.labnetinternational.com/products/biopette-plus-autoclavable-multichannel-pipettes>.
Leinco Technologies, Inc. Immunohistochemistry Protocol for Frozen Sections. <http://www.leinco.com/immunohistochemistry>.
ThermoFisher. Alexa Fluor 488 Phalloidin. <https://www.thermofisher.com/order/catalog/product/A12379>.
Real Time PCR is a method that is often used for analysis of RNA samples, since standard PCR does not really work with RNA itself, and doing the PCR analysis in real time instead of with a gel afterwards provides quantitative data that can be later manipulated with statistics - again, another use of Excel. Since the starting material is RNA, it must first be converted to cDNA through a reverse transcription process before it can be used for real time PCR. Real time PCR utilizes a machine that can monitor the polymerase chain reaction as it happens by noting when it sees the samples fluorece as the cDNA has progressed through another cycle. For our machine we must use 384 well plates, which have very small wells and can be a bit disorienting at first. Thankfully, our lab has a few multichannel pipettes which makes it so we can load multiple sample at once. Real time PCR seemed like a daunting task to me before I had tried it, but once I had I realized it was not as difficult as I thought it would be. The advice that the graduate student in my lab gave me was that the hardest part is simply remembering your place on the plate, and I feel like that is true for most procedures where you have to load samples, though it is a bit harder since there are so many more wells for this than ever would be for a standard PCR or a Western Blot.
 |
| Figure 1. 384 well plate with labels (E&K Scientific). |
 |
| Figure 2. Multichannel pipettes (Labnet). |
Immunohistochemistry is one of those lab procedures that not only can provide important information about the morphology of cells, but is also quite fascinating and reminds me how science can allow us to look at parts of our world that we normally cannot. During many of the Friday lunch lectures that the mentors have been giving, they have been excitedly showing us pictures of cells that have been stained and imaged - even people who have their Ph.D.s already and have been looking at cells for years are still amazed by the beauty of these pictures. There are many different dyes that can be used for immunohistochemistry, and the the fluorescent dyes give some of the most interesting pictures. Two are shown in below: the DAPI fluorescent dye stains the nuclei of cells, whereas phalloidin 448 stains the actin and the cytoskeleton (ThermoFisher).
 |
| Figure 3. Example of immunohistochemistry on HEK 293 cells, using fluorescent dyes DAPI, blue, and phalloidin 448, green (Bruce, 2013). |
The protocol for immunohistochemistry was explained to me initially by the technician I work with in regards to its is similarity to Western Blotting, since it requires the use of a primary and secondary antibody. The overall procedure combines many different techniques, as first cells must be grown and treated appropriately, then cover slips have to be probed so they can imaged under a microscope that is attached to a computer that allows pictures to be taken and distributed.
 |
| Figure 4. A schematic of how the primary and secondary* antibody allow visualization of the desired proteins (Leinco Technologies, Inc). *secondary antibody is labeled here as "fluorescent tag," which is also accurate |
I've again been having issues, this time with my cells. A few weeks ago, I had frozen down cells to store them and have been plating them for my project. The first time I plated them everything went fine, and I continued to grow them for a bit. One of the later times when I was splitting my cells, I believe that I did not do it aggressively enough and when I checked the dishes later under the microscope, I looked around the entire plate and could find no cells. I had been leaving plates in the incubator with only media in them as I must have left the cells in the plate I discarded when trying to split them. However, I was not incredibly concerned because I still had more aliquots of my frozen down cells, and I simply plated one of those. When I checked those cells, they were absolutely dead. I tried plating more cells, and they were also dead. Next week I will be attempting to plate cells again, using aliquots that someone else had froze down in case an issue occurred when I froze the cells down. Something could also be going wrong as I am plating the cells, but overall I just hope that the cells will adhere to the bottom of the plate and grow as they should, as I need them to complete the next tasks for my project.
References:
Bruce, D. 2013. Cell culture and stem cell techniques. <http://www.laney.edu/wp/doug_bruce/cell_culture_stem_cell_training/>.
E&K Scientific. 384 Well Roche 480 Plate, White, Barcoded. <http://www.eandkscientific.com/384-Well-PCR-Plate-White-for-Roche-Light-Cycler-Barcoded.html>.
Labnet. BioPette Plus Autoclavable Multichannel Pipettes. <http://northamerica.labnetinternational.com/products/biopette-plus-autoclavable-multichannel-pipettes>.
Leinco Technologies, Inc. Immunohistochemistry Protocol for Frozen Sections. <http://www.leinco.com/immunohistochemistry>.
ThermoFisher. Alexa Fluor 488 Phalloidin. <https://www.thermofisher.com/order/catalog/product/A12379>.
Week Six
Our Monday meeting was pushed to Tuesday so we could take off Independence Day if we wanted to. I took most of the day off, but I had to come in for a short time to split the cells I had been working on since they were starting to become quite confluent and I was going to have to split them for another drug trial anyways. However, the concentrations were way too low that day so I split the cells normally so they could grow another day and hopefully when I checked again, the concentrations would be what I needed. I was excited to get ahead with my trial, but with the point that the cells were at, the overall protocol would be done by Friday even with another day of waiting for the cells to reach the needed concentration. However, it was definitely not the start to the week that I had wanted.
Small issues continued to appear and hinder me from being able to complete tasks on the days I planned to, but by the end of the week I was able to get the results I was aiming to and that's what really matters. In our lab, we make the gels for our Western Blots - they are available for purchase precast, but as many lab materials, they are quite expensive and we run Western Blots quite often. Below is a video that accurately explains how to make the two gel layers, in which the exact reagent amounts will depend on the percentage gel desired (labtricks, 2010).
When I went to take the comb out most of the wells were not present and others had merged with the wells next to them, so there was no way I could load the gel and run it with no where to put the samples - this simple issue set my Western Blot back a day. Again, I was still set to be finished by Friday, but it was nevertheless frustrating that my plans were changed outside of my control. However, problems that come up during lab work must be taken in stride, and often can make you a better scientist as you then must go back and reevaluate your techniques or the theory behind the procedure to fix what was wrong.
I was shown how to quantify the bands from the Western Blot on the computer so I could more precisely compare them. The program we used to analyze the raw quantification data was Microsoft Excel, just the same as is used in most laboratory courses. I figured as I learned how to use Excel in lab classes that I would need to have the skills to use it in the future as it is a helpful tool for organizing data and streamlining calculations, and it was nice to see the skills I learned were applicable in a practical setting outside of the classroom. Once I had quantified my Western Blot, my results gave me guidance on what I would need to do for the next week and allowed me to analyze how effective the drug had been.
Every month, a large group of the people who work on my floor get together in our common room and we have a potluck. I was able to participate in the June potluck before, but I simply bought salsa and chips. However, this month we were having an Independence Day themed potluck on July 5th when everyone would be back, and I decided to make pasta salad as I felt it fit the theme since I had often encountered various types of pasta salad at summer cookouts. One of the women who works on my floor brought her daughter to the potluck and later in the week told me me that most of what she ate was my pasta salad, which really made my day. I honestly have never enjoyed cooking that much - I prefer baking - so it was encouraging to hear that people liked what I had made. I have also found myself more inclined to cook since I had started in the lab because cooking in many ways is so similar to the systematic approach that is required for the lab work I enjoy, and also gives me a meal in the end.
Small issues continued to appear and hinder me from being able to complete tasks on the days I planned to, but by the end of the week I was able to get the results I was aiming to and that's what really matters. In our lab, we make the gels for our Western Blots - they are available for purchase precast, but as many lab materials, they are quite expensive and we run Western Blots quite often. Below is a video that accurately explains how to make the two gel layers, in which the exact reagent amounts will depend on the percentage gel desired (labtricks, 2010).
When I went to take the comb out most of the wells were not present and others had merged with the wells next to them, so there was no way I could load the gel and run it with no where to put the samples - this simple issue set my Western Blot back a day. Again, I was still set to be finished by Friday, but it was nevertheless frustrating that my plans were changed outside of my control. However, problems that come up during lab work must be taken in stride, and often can make you a better scientist as you then must go back and reevaluate your techniques or the theory behind the procedure to fix what was wrong.
 |
| Figure 1. My representation of what a Western Blot gel with all of its wells after the comb has been removed looks like versus what my gel looked like when I removed the comb. |
Every month, a large group of the people who work on my floor get together in our common room and we have a potluck. I was able to participate in the June potluck before, but I simply bought salsa and chips. However, this month we were having an Independence Day themed potluck on July 5th when everyone would be back, and I decided to make pasta salad as I felt it fit the theme since I had often encountered various types of pasta salad at summer cookouts. One of the women who works on my floor brought her daughter to the potluck and later in the week told me me that most of what she ate was my pasta salad, which really made my day. I honestly have never enjoyed cooking that much - I prefer baking - so it was encouraging to hear that people liked what I had made. I have also found myself more inclined to cook since I had started in the lab because cooking in many ways is so similar to the systematic approach that is required for the lab work I enjoy, and also gives me a meal in the end.
 |
| Figure 2. An accurate depiction of my thoughts as I have been working in the lab (Sketching Science, 2016). |
References:
labtricks. 2010. How to Make an SDS-Page gel. <https://www.youtube.com/watch?v=EDi_n_0NiF4>.
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