October 26, 2011
This was my favorite Wednesday so far. Today we did cell culture. JM decided that SeungHa and I would have separate turns, so we could have one on one. I was able to go first. He guided us through each step throughout the whole process. The point of this project was to be able to count the number of cells that we were making. I started by pipetting 10ml of medium, aspirating it out, and repeating this process a couple of times. Then, I pipetted 2ml of Typsin and gently rocked the container back and forth to make a layer on the bottom of the container. Then we placed the container into an incubator for 5 minutes. Afterwards, we pipetted 8ml of medium into the container and placed it in a centrifuge which spins it around really quickly. The spinning created a separation between the medium and the cells. We then aspirated the medium but not the cells. We pipetted 10ml of medium into the container that only contained the cells. Then we pipetted two 10ml samples into a small graduated pipet and added Trypin Blue. Trypin Blue indicates dead cells under a microscople. After this whole procedure, we went downstaris and looked at the cells that I made under their high-tech microscope. They have a certain machine that allows us to look at a certain area of cells. We manually counted these cells in this area and multiply it by a very big number which I don't remember. That number is an approximation of the total number of cells. Then we brought the cells to a different machine that automatically counted the cells. Once we finished, SeungHa repeated the same process that I did.
Sunday, October 30, 2011
Tuesday, October 25, 2011
Day 10
October 26, 2011
We didn't do too much today. The whole time we made polyacrylamide. I'm not exactly sure why we made it and what its purpose was but it came out as a gelatin. Kellin just guided me through each step. To make the gelatin, we mixed acrylamide with bisacrylamide. The acrylamide was 12.5% weight per volume. There were two different bisacrylamides. One was 1.3% bisacrylamide weight/volume. The other was 0.15% bisacrylamide weight/volume. The 1.3% would end up as a harder gelatin while the 0.15 would end up softer. Before we mixed the acrylamide and bisacrylamide, we had to create a solution with a 4 to 1 ratio of the substances in order for it to work. So we used 40ml of acrylamide and 10ml of bisacrylamide. Then we added 2ml of a chemical called Temed to each of the mixtures. Temed makes the reaction undergo. But for some reason, it smells very badly. It had a pungent stench that smelled like rotton fish. So we had to make sure to open and close the bottle very quickly! Then we added 5ml of 10% APS. We had to wait for about 10 minutes for the gelatin to form. After waiting, he took the gelatin out of the container and let me hold them. It was really cool because it felt exactly like jello. The hard gelatin formed perfectly, but the softer gelatin came out too soft. So then we tried the whole process again but making a larger amount of polyacrylamide. This time, the soft gelatin formed right, but the hard gelatin did not. Kellin realized that the problem may be that the acylamide might be too old. He made a new acrylomide, but we did not have enough time to perform the experiment again.
We didn't do too much today. The whole time we made polyacrylamide. I'm not exactly sure why we made it and what its purpose was but it came out as a gelatin. Kellin just guided me through each step. To make the gelatin, we mixed acrylamide with bisacrylamide. The acrylamide was 12.5% weight per volume. There were two different bisacrylamides. One was 1.3% bisacrylamide weight/volume. The other was 0.15% bisacrylamide weight/volume. The 1.3% would end up as a harder gelatin while the 0.15 would end up softer. Before we mixed the acrylamide and bisacrylamide, we had to create a solution with a 4 to 1 ratio of the substances in order for it to work. So we used 40ml of acrylamide and 10ml of bisacrylamide. Then we added 2ml of a chemical called Temed to each of the mixtures. Temed makes the reaction undergo. But for some reason, it smells very badly. It had a pungent stench that smelled like rotton fish. So we had to make sure to open and close the bottle very quickly! Then we added 5ml of 10% APS. We had to wait for about 10 minutes for the gelatin to form. After waiting, he took the gelatin out of the container and let me hold them. It was really cool because it felt exactly like jello. The hard gelatin formed perfectly, but the softer gelatin came out too soft. So then we tried the whole process again but making a larger amount of polyacrylamide. This time, the soft gelatin formed right, but the hard gelatin did not. Kellin realized that the problem may be that the acylamide might be too old. He made a new acrylomide, but we did not have enough time to perform the experiment again.
Sunday, October 23, 2011
Day 9
October 19, 2011
Today was an uneventful day. We first watched JM do an experiment where he made different shaped cells. He said that the dishes needed to be washed out several times before putting the cells in just to be extra cautious. Each time the dishes were washed, we had to wait a few minutes before he could wash them again. I think he had to wash them 4 times. He then separated the cells from the media. In order to do that, he added trypsin which is a chemical that cuts proteins into small pieces so that the cells are free from the media. He attached fluorescent molecules to the particles so that different areas of the particles would be indicated under a microscope. He was afraid that the fluorescent molecules would not work because they usually only last up to one year, but it was 5 years old. So then of course we went downstairs for him to look at the particles under the microscope. We were there for about an hour for him to focus the microscope on the different cells. We looked at sphere, rod, and worm cells. The red fluorescence indicated the location of the nanoparticles. The blue showed the Nuclei. And the green indicated the lysosomes. He planned for us to do cell culture but we ran out of time, so he said that we would do it next time.
Today was an uneventful day. We first watched JM do an experiment where he made different shaped cells. He said that the dishes needed to be washed out several times before putting the cells in just to be extra cautious. Each time the dishes were washed, we had to wait a few minutes before he could wash them again. I think he had to wash them 4 times. He then separated the cells from the media. In order to do that, he added trypsin which is a chemical that cuts proteins into small pieces so that the cells are free from the media. He attached fluorescent molecules to the particles so that different areas of the particles would be indicated under a microscope. He was afraid that the fluorescent molecules would not work because they usually only last up to one year, but it was 5 years old. So then of course we went downstairs for him to look at the particles under the microscope. We were there for about an hour for him to focus the microscope on the different cells. We looked at sphere, rod, and worm cells. The red fluorescence indicated the location of the nanoparticles. The blue showed the Nuclei. And the green indicated the lysosomes. He planned for us to do cell culture but we ran out of time, so he said that we would do it next time.
Wednesday, October 19, 2011
Day 8
October 18, 2011
Today, we did the same lab as last time but added a few more things. We made several different standards of Gel-MA; 0.5ml,1ml, 5ml, 10ml, and 20ml. Each required the same process but differ in amount. The process did not consist of many steps. The hardest part was the first part; filling the channel with the Gel-MA. It took both of us a few tries to get it, but we both were able to do it eventually. It was difficult because I have to inject the Gel-MA into the tiny channel using a pipette. If I inject it too quickly, it will cause the formation of air bubbles, and then I have to start the whole process over. Every time that I mess up, I have to clean the channel with ethanol before starting over. Last Tuesday, it took my at least 5 attempts to finally get it, but today it only took me 3 tries! Then, we placed 100 microliters over the outlet of the channel. Then, depending on which standard we were making, we put a certain amount of protein solution over the inlet. We then waited for 8 minutes for the standards to settle. Once all of the standards were created, we walked over to a different building in order to have a closer look at the standards. We put them in a typhoon which scans over cells and measures the amount of fluorescence. The typhoon takes approximately 13 minutes to scan the cells. The results showed up on a computer attached to the typhoon. The more concentrated ones were darker while the less concentrated ones appeared lighter on the computer. By this point, it was about 4:20, so we had to speed walk back to the other building so I could get back in time!
Sunday, October 16, 2011
Day 7
October 12, 2011
Earlier in the day, the mentor added particles to cells for us to use when we arrived. Once we came, we took the cells and brought them to lab on the floor below our lab. He placed the cells under a fluorescent microscope. It took him about an hour to have the perfect focus and angle. When it was all set up, we were able to see the different shaped cells. We first saw the sphere cells. Those looked smaller than any of the other ones. They were about 50nm each. Then we saw rod cells. Those were a little more rotund but also a little longer which were about 150nm each. The last kind that we saw were the worm cells. Those were about the same width as the rods but were even longer, and they were 250nm long. In order for us to be able to see the particles under the microscope, particular molecules had to be attached to them. These molecules allowed the particles to show up under a red fluorescent light. There are different colored lights for different molecules. There are particular molecules that let particles show up under either green or blue fluorescent lights. After we looked at the particles, we headed back upstairs. Then, JM printed out a sheet for us that showed us the different shapes of the particles at an even closer look. This was pretty much all that we did today.
Earlier in the day, the mentor added particles to cells for us to use when we arrived. Once we came, we took the cells and brought them to lab on the floor below our lab. He placed the cells under a fluorescent microscope. It took him about an hour to have the perfect focus and angle. When it was all set up, we were able to see the different shaped cells. We first saw the sphere cells. Those looked smaller than any of the other ones. They were about 50nm each. Then we saw rod cells. Those were a little more rotund but also a little longer which were about 150nm each. The last kind that we saw were the worm cells. Those were about the same width as the rods but were even longer, and they were 250nm long. In order for us to be able to see the particles under the microscope, particular molecules had to be attached to them. These molecules allowed the particles to show up under a red fluorescent light. There are different colored lights for different molecules. There are particular molecules that let particles show up under either green or blue fluorescent lights. After we looked at the particles, we headed back upstairs. Then, JM printed out a sheet for us that showed us the different shapes of the particles at an even closer look. This was pretty much all that we did today.
Day 6
October 11, 2011
Today was my first day at my real project. It was exciting to finally see what I was going to be doing. The building that I am in on Tuesdays, the Smith building, is a lot newer than Maryland Hall, and it was very obvious as soon as I walked in. The building was 10 times nicer and twice the size than Maryland Hall. Once we arrived at the lab, there were multiple graduate students working in there as well. The lab was also much nicer and larger. My mentor, Kellin, started by explaining again what my project was. In short, I will be looking at cut nerves, and trying to figure out a way to reconnect one side to the other. In order to do this, there are two options. One way is to use electro spun fibers. The second way is to make a gradient. I will be focusing on making a gradient. When making a gradient, the goal is to be able to move protein from one side to the other. So if the protein is attracted to the cell, then it will want to move from a lower concentration to a higher concentration. And if the protein repels the cell, then it will want to move from a higher concentration to a lower concentration. So we took 5% w/v Gel Ma. It contains 0.5% Irgacure which allows MA groups to react. Then we made 20 micrograms/ml of GDNF from 3 microliters of 833 micrograms/ml. To figure out all of the calculations, we needed to do stoichiometry. Then we used a PDMS strip with a channel and placed the Gel MA into the channel. On top of one side of the strip, we put a big drop of 20 micrograms/ml GDNF. And on the other side of the strip, we put a little drop. The point of this was to make a gradient with a higher concentration on one side and a lower concentration on the other. The first time we did this, he did the whole thing and I watched. Then for the second time, he let me do everything, and he just guided me through the steps. It took me a few tries to get everything right, but he was very patient about it. Next Tuesday, we will look at the gradient we made under their high-tech microscope.
Sunday, October 9, 2011
Day 5
Wednesday October 5, 2011
Today was the most interesting day so far. We started by finishing our nanoparticle project from yesterday. While finishing up, we learned more in depth about nanoparticles. Nanoparticles are positive charged because it helps when interacting with the cell which is negatively charge. The more positively charged the particles are, the easier the interaction becomes. Also, the size of the nanoparticles is important. Only a certain size can fit through the cell membrane which is less than 200 nanometers. Ours were about 1,000 nanometers which was a little too big. Once we finished that project, we watched our mentor do a lab. He kept using this machine that precisely adds water to a solution to dilute it to the desired amount. It was also known as a syringe pump. However, he tried this process several times, but for some reason, the machine wasn't working. Next time, we will get to see what shape the cells formed. Afterwards, our mentor, JM, showed us different lab journal and explained how to properly keep one. He showed us examples of graduate students' journals and under graduate students' journals. Surprisingly, the under grad. students' journals tended to be neater. Then, JM introduced us to this website called PubMed. This website had thousands of different science related journals on almost every topic imaginable. It was really outstanding. Next week, I will be going with my real mentor for the first time on Tuesday. I’m excited to start my real project!
Thursday, October 6, 2011
Day 4
October 4, 2011
Today I was with Seung Ha again. Starting next week, I will go with my mentor on Tuesdays. So, when we arrived, her mentor left us a note saying that he had a class until 2pm and to read the packets that he provided. So Seung Ha and I walked to the library to read the packets there. But we got lost on both the way there and the way back. Quite the experience. Anyways, not too much happened today. We were still mostly learning basic skills and going over the introduction to the lab. We learned about stoichiometry and when the conversions will need to be made. Sometime around 3pm, my mentor, Kellin, showed up for a little while just to check in before heading to his next class. Then, we made nanoparticles. We pipetted water with the polymer and DNA. Afterwards, we put the mixture into a machine that shoots light through the mixture and determines the size of the particles. The size of the particles matter because the cell only takes in nanoparticles when they are a certain size. But before each of these processes, there was some waiting time. In the meantime, we watched her mentor perform an experiment that he needed to do for class. He explained his project to us, but it was still hard to understand what he was doing. This was about all we did today.
Sunday, October 2, 2011
Day 3
September 28, 2011
Today was our first real day! We arrived around 1:30 and we each went to our different labs. Since today is Wednesday, I went with Seung Ha to her lab in Maryland Hall. When we arrived, her mentor first gave us an introduction and explained the project to us again. He then drew it out for us so we would have a better understanding. Then, we moved to the lab room. He explained the rules of the lab room. Afterwards, we practiced pipetting, a skill that we would need to have in order for our labs to be accurate. We practiced different techniques for the first two hours. We tried the normal pipetting technique with different amounts of water for the first hour. Then we tried reverse pipetting for the second hour. John-Michael showed us how to pipette in the most accurate way. He told us that it was important that no air bubbles are made while pipetting because it will make the reading inaccurate. Once Seung Ha and I understood all we needed to know about pipetting, John-Michael showed us cells that were in the process of being grown. He pointed out how all the different cells are different sizes and different shapes because of their structure. The cells are kept in a special machine. I forget what the machine is called but it sort of looks like a refrigerator. This machine makes sure that no outside sources interrupt the growth of the cells. Then, we looked at a poster about the Preparation of Ternary Nanoparticles that John-Michael made for class. He explained how a polymer was made of multiple monomers and the importance of plasmid DNA. It was all pretty hard to understand because he is learning graduate material. But from what I did understand, it was interesting. For the rest of the time, he showed us around the building. We went to other labs where he explained the significance of different tools that we might use sometime. Sometime near the end of the day, my mentor showed up for a few minutes before he needed to leave for his next class. He came in and checked to see what we were up to. He then explained my project to me again in a little more detail. Then he had to leave for he wasn’t late for his next class. Since it was only our first day, that was pretty much all that we did!
Day 2: Luncheon
On Tuesday, we finally met our mentors for the first time. My mentor's name is Kellin Krick, and he is a graduate. He grew up in Ohio which is also where he went to college. He decided that he wanted to be a biologist in his junior year in high school. When I found out my project the night before, I was very surprised and shocked, but since my mentor seems very nice and patient, I am a little more eager to start than I was before. While we ate lunch, he briefly described what my project was in more detail. Even though it is still pretty hard for me to understand, I think that I am going to be regenerating nerves. I am going to learn how to fill the nerve with the right amount of gradient so there is a balance in both sides of the nerve. We will be working on the Bio-medical campus which is about 10 minutes away from the main campus. Kellin said that he would drive me to the Bio-Medical campus. The only problem is that he is only available on Tuesdays because he has a class Wednesday afternoons. So, I will be with Seung Ha and her mentor, John-Michael, on Wednesdays helping them with her project. She is looking at cancer cells and determining the missing gene. Then, she is going to try to place that gene back into the cell so it isn't cancerous anymore. So I guess I have two projects! I think that the hardest part for me will be keeping up with Seung Ha. Since she is doing the same project both days, I will only receive half of the information that she will learn.
When we finished eating, we filled out a survey, took a picture for our Hopkins’s ID's, and then walked around the campus to find which lab each of us would be located in. I was only able to see the lab I would be in on Wednesdays with Seung Ha. It is room 138 in Maryland Hall. It is a condensed lab with multiple rooms within it. The center room is the main lab where we will be performing experiments. Some of the other rooms are only for brainstorming. There are no lab materials, only desks and chairs. The last room is where unfinished labs were kept. Once we looked around for a little bit, we finally headed back to Garrison.
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