November 29, 2011
I didn't start until pretty late today because he had a long class today. But once we started we once again made a gel but we used a wider channel. I wish I could find a picture of the channel to show you so you could have a better understanding of what the channel does and how it works. Kellin wanted to try to make a wider gel to see if it would make a better gradient. Since we were trying to make a wider gel, we obviously had to use more solution to make it. We took the whole process a whole step further today and actually electrospun the gel. Electrospinning is when polymers are spurt onto the gel to make fibers which is stuck on this very rapidly spinning wheel. The reason for this is to try to align all the fibers to be going straight up and down the gel. The purpose of aligning them straight is that once the gradient is placed in the rat, the proteins will moved directly through the gradient from one side of the nerve to the other. When he first tried it, it didn't work because he accidently used the wrong polymer to make the fibers. The polymer was too viscous to be able to be electrospun. Then he tried again with the correct polymer and it did work! He still had to check under the microscope though to se if the fibers were aligned correctly. Surprisingly, they were aligned pretty straight even though it was the first trial. He felt pretty proud of himself!
Tuesday, November 29, 2011
Sunday, November 20, 2011
Day 17
November 16, 2011
Today we learned all about Gel elctrophoresis! This technique is used whenever scientists want to sort DNA according to their size. Before we arrived, JM made gel for us to use in the experiment. We placed the gel into a container which was used as a filter that sorts DNA strands. We made many small holes in the gel which was where we would place the DNA strands. We injected 12 microliters of DNA strands each hole. An electrical current was added to the gel and DNA strands. The current caused the DNA strands to move across the gel, but the shorter strands moved faster. This happened for all the different sizes of DNA. The problem was that it takes multiple hours for the electrophoresis to finish so we weren't able to see the final result. While we were waiting for the electrophoresis, we brought some cells that he previously made to the microscope. However, he wasn't able to see the particles for some reason. He assumed that it was because the particles was so small. It was strange though because the particles were attached to fluroescent particles so there should have been no reason why he couldn't see the fluorescent colors. So then he tried putting non fluorescent cells that he hadn't made under the microscope to see if he could see these ones. If that didn't work, that meant that the fluorescent dye was not responsive as opposed to there being a problem with the batch of cells he had made. Luckily, the non fluorescent cells were not visible either so he knew that it was just the fluorescent particles that were not working. It is kind of interesting when he works with the microscope but the only problem is that we aren't able to do anything because the equipment is really expensive and complex and he wouldn't want us to mess anything up. So usually this isn't my favorite thing to do because it also takes a long time for him to focus the microscope correctly while we just have to sit there. But the day did get better once he finished that. I asked if we could go over the protocol again for the electrophoresis just so I had a better understanding. He found this interactive lab online that explained everything step by step. It was so helpful and was fun to do. I was actually just on the website again to help me write my blog!
Today we learned all about Gel elctrophoresis! This technique is used whenever scientists want to sort DNA according to their size. Before we arrived, JM made gel for us to use in the experiment. We placed the gel into a container which was used as a filter that sorts DNA strands. We made many small holes in the gel which was where we would place the DNA strands. We injected 12 microliters of DNA strands each hole. An electrical current was added to the gel and DNA strands. The current caused the DNA strands to move across the gel, but the shorter strands moved faster. This happened for all the different sizes of DNA. The problem was that it takes multiple hours for the electrophoresis to finish so we weren't able to see the final result. While we were waiting for the electrophoresis, we brought some cells that he previously made to the microscope. However, he wasn't able to see the particles for some reason. He assumed that it was because the particles was so small. It was strange though because the particles were attached to fluroescent particles so there should have been no reason why he couldn't see the fluorescent colors. So then he tried putting non fluorescent cells that he hadn't made under the microscope to see if he could see these ones. If that didn't work, that meant that the fluorescent dye was not responsive as opposed to there being a problem with the batch of cells he had made. Luckily, the non fluorescent cells were not visible either so he knew that it was just the fluorescent particles that were not working. It is kind of interesting when he works with the microscope but the only problem is that we aren't able to do anything because the equipment is really expensive and complex and he wouldn't want us to mess anything up. So usually this isn't my favorite thing to do because it also takes a long time for him to focus the microscope correctly while we just have to sit there. But the day did get better once he finished that. I asked if we could go over the protocol again for the electrophoresis just so I had a better understanding. He found this interactive lab online that explained everything step by step. It was so helpful and was fun to do. I was actually just on the website again to help me write my blog!
Wednesday, November 16, 2011
Day 16
November 15, 2011
Seungha came with me today. Both of our mentors had a seminar to go to. The reason why both of our mentors were going was because Dr. Mao, our professor, invited the speaker, so they were pretty much obligated to go. So we were also able to listen in on the seminar. But as soon as the speaker projected the first slide, "Design of Self-Assembled Peptide Hydrogels For Delivery", I was already completely lost before the seminar even started. I really tried to listen and understand, but it there were too many terms that I didn't understand. I did get a few things out of it though. The speaker's job is to manipulate peptide structures. There are two different ways in doing this. The first is doing media based injections. However, this process is really inefficient which is why they don't focus on it. They use the technique of employing phase transition materials. I'm not exactly sure what this means, but I know that this was focus of the seminar. He said that they initially failed when trying this technique and had to try several different trials. They finally figured out the most efficient way. They would design a family of peptides which were highly soluble. The solubility allowed the peptides to be injectable. They've designed between 50 and 60 kinds of peptides so far. One peptide that he focused on was named Max 1. Max 1 is composed of hydrophylic and hydrophobic cells. The significance of this peptide is its ability to self-assemble. When placed in 5 degrees celsius, it unfolds. But when the temperature is raised to 40 degrees celsius, it reassembles. I'm not sure the importance of "self-assembly" but they all seemed to think it is. They he showed some pictures of mice and rats being injected with Max 1. I seemed to be the only one who found the pictures sad and disturbing while everyone else found them fascinating. From then on, I couldn't understand anymore because the speaker went into many more details that completely made no sense. Once the seminar was over it was around 4:00 so they let us go a little early.
Seungha came with me today. Both of our mentors had a seminar to go to. The reason why both of our mentors were going was because Dr. Mao, our professor, invited the speaker, so they were pretty much obligated to go. So we were also able to listen in on the seminar. But as soon as the speaker projected the first slide, "Design of Self-Assembled Peptide Hydrogels For Delivery", I was already completely lost before the seminar even started. I really tried to listen and understand, but it there were too many terms that I didn't understand. I did get a few things out of it though. The speaker's job is to manipulate peptide structures. There are two different ways in doing this. The first is doing media based injections. However, this process is really inefficient which is why they don't focus on it. They use the technique of employing phase transition materials. I'm not exactly sure what this means, but I know that this was focus of the seminar. He said that they initially failed when trying this technique and had to try several different trials. They finally figured out the most efficient way. They would design a family of peptides which were highly soluble. The solubility allowed the peptides to be injectable. They've designed between 50 and 60 kinds of peptides so far. One peptide that he focused on was named Max 1. Max 1 is composed of hydrophylic and hydrophobic cells. The significance of this peptide is its ability to self-assemble. When placed in 5 degrees celsius, it unfolds. But when the temperature is raised to 40 degrees celsius, it reassembles. I'm not sure the importance of "self-assembly" but they all seemed to think it is. They he showed some pictures of mice and rats being injected with Max 1. I seemed to be the only one who found the pictures sad and disturbing while everyone else found them fascinating. From then on, I couldn't understand anymore because the speaker went into many more details that completely made no sense. Once the seminar was over it was around 4:00 so they let us go a little early.
Sunday, November 13, 2011
Day 15
November 9, 2011
Today we made more cells. We pipetted 20 microliters of PBS into 4 rows with 6 slots in each row. Then we added 20 microliters of BSA which is a standard protein to the PBS. With the mixture of PBS and BSA, we diluted it several times so there was very little of each chemical by the end slot. We added Cu+2 which changed the clear protein molecules to a light green color. When the Cu+2 was added to the protein, it became Cu+1. Then we added BCA. Two BCAs bind with one Cu+1 which turns the mixture purple. We used this machine to determine how much light is absorbed in each slot. However, according to JM, the numbers seemed to be lower than he expected. He assumed that it was because the cells were frozen for a long time beforehand. Afterwards, JM showed us the way to sterylize pipette tips. This technique is called autoclaving. He placed the tips in a very large machine that pretty much cooks the pipette tips until all of the germs are killed. The machine gets VERY hot. He needed to use gloves when putting in the tips and taking them out. He also wouldn't allow us to stand in front of the machine just in case a big amount of smoke came out. At the end of the day, he explained to us the process of freezing down cells for long term storage. When thawing the cells, he has to go through several steps to slowly cool them down so the cells don't die from a sudden change in temperature. My favorite part today was watching the cells color change of the and learning why they did change. However, it was tough to understand the whole process with all of the different proteins. I tried asking about the significance of each protein like BCA, BSA, and PBS. He really tried to explain it to us but it was very difficult for him to decribe each of them. That it why it was a little hard for me to write this blog because I don't exactly understand what each protein is.
Today we made more cells. We pipetted 20 microliters of PBS into 4 rows with 6 slots in each row. Then we added 20 microliters of BSA which is a standard protein to the PBS. With the mixture of PBS and BSA, we diluted it several times so there was very little of each chemical by the end slot. We added Cu+2 which changed the clear protein molecules to a light green color. When the Cu+2 was added to the protein, it became Cu+1. Then we added BCA. Two BCAs bind with one Cu+1 which turns the mixture purple. We used this machine to determine how much light is absorbed in each slot. However, according to JM, the numbers seemed to be lower than he expected. He assumed that it was because the cells were frozen for a long time beforehand. Afterwards, JM showed us the way to sterylize pipette tips. This technique is called autoclaving. He placed the tips in a very large machine that pretty much cooks the pipette tips until all of the germs are killed. The machine gets VERY hot. He needed to use gloves when putting in the tips and taking them out. He also wouldn't allow us to stand in front of the machine just in case a big amount of smoke came out. At the end of the day, he explained to us the process of freezing down cells for long term storage. When thawing the cells, he has to go through several steps to slowly cool them down so the cells don't die from a sudden change in temperature. My favorite part today was watching the cells color change of the and learning why they did change. However, it was tough to understand the whole process with all of the different proteins. I tried asking about the significance of each protein like BCA, BSA, and PBS. He really tried to explain it to us but it was very difficult for him to decribe each of them. That it why it was a little hard for me to write this blog because I don't exactly understand what each protein is.
Thursday, November 10, 2011
Day 14
November 8, 2011
The goal for today was to create a non-linear gradient. Kellin had never tried making this before, so it was going to be an interesting experience. It was the same process as what I did the other days just with different volumes. We did two different trials. In the first one, we added 5 microliters of the mixture of gelatin and protein onto the inlet of the channel every minute. In the second trial, we added 5 microliters onto the inlet adding 2 minutes on the previous time. For instance, we first started with waiting for 2 minutes. After 2 minutes, we then added 5 more microliters and then waited for 4 minutes. Meanwhile, I asked about the purpose of the channel and gradient and what it was for. Kellin explained that once the gradient was made, they will electrospin fibers onto it in a way that the fibers are aligned parallel inside the gradient. Then they will wrap the gradient around a wire. Then a few days later they will peel it off from the wire and the gradient will be cylindrical. If it works, they'll inject the gradient into a rat to test it. They will cut open the leg of a rat and cut the nerve. Then they will place the gradient there and stitch up the leg. For a few days, the rat won't be able to move that leg, but the goal is that the nerve will regrow through the gradient and reattach itself. By the time he finished explaining, the gradients were both done. We walked over to a different hospital about 5 or 10 minutes away to use this machine called a typhoon. The typhoon measures the concentration and structure of gradients. We saw that one of the gradients just didn't work at all, and the other gradient was closer to linear than nonlinear. Even though we ended up failing pretty badly, it was a good experience for us to learn from our mistakes.
The goal for today was to create a non-linear gradient. Kellin had never tried making this before, so it was going to be an interesting experience. It was the same process as what I did the other days just with different volumes. We did two different trials. In the first one, we added 5 microliters of the mixture of gelatin and protein onto the inlet of the channel every minute. In the second trial, we added 5 microliters onto the inlet adding 2 minutes on the previous time. For instance, we first started with waiting for 2 minutes. After 2 minutes, we then added 5 more microliters and then waited for 4 minutes. Meanwhile, I asked about the purpose of the channel and gradient and what it was for. Kellin explained that once the gradient was made, they will electrospin fibers onto it in a way that the fibers are aligned parallel inside the gradient. Then they will wrap the gradient around a wire. Then a few days later they will peel it off from the wire and the gradient will be cylindrical. If it works, they'll inject the gradient into a rat to test it. They will cut open the leg of a rat and cut the nerve. Then they will place the gradient there and stitch up the leg. For a few days, the rat won't be able to move that leg, but the goal is that the nerve will regrow through the gradient and reattach itself. By the time he finished explaining, the gradients were both done. We walked over to a different hospital about 5 or 10 minutes away to use this machine called a typhoon. The typhoon measures the concentration and structure of gradients. We saw that one of the gradients just didn't work at all, and the other gradient was closer to linear than nonlinear. Even though we ended up failing pretty badly, it was a good experience for us to learn from our mistakes.
Wednesday, November 9, 2011
Day 13
November 2, 2011
Today we did really complicated things that I'm not actually sure how to explain. We talked about n/p charges (negative to positive) which was what our experiment concerned. The goal of the experiment was to see an increase of protein. We begun by taking Lucifase Assay Substrate (LAS) and placing 100 microliters in 4 different tubes. Then we took 200 microliters of cell lyate and placed it in a 4x6 plate. Cell lyate were the proteins from the cells. Then we mixed the LAS with the cell lyate. We then placed the tubes into this machine that determined the n/p ratio. Meanwhile, JM used excel to plot the data. The data ranged anywhere in between 5 million and 12 million cells for the different trials. We were looking for an increase of protein as the trials went on. And for once, we got a good result! The number of proteins did end up increasing. Then we repeated all of these steps five times to make sure that we obtained accurate data. Seung Ha and I both got turns doing this process. However, our data wasn't accurate because we produced a few bubbles when we initially pipetted which throw off the results. Then, JM taught us how to plot the data that we received. The process doesn't sound hard, but it was very tedious and took about the whole time. So this was it for today!
Today we did really complicated things that I'm not actually sure how to explain. We talked about n/p charges (negative to positive) which was what our experiment concerned. The goal of the experiment was to see an increase of protein. We begun by taking Lucifase Assay Substrate (LAS) and placing 100 microliters in 4 different tubes. Then we took 200 microliters of cell lyate and placed it in a 4x6 plate. Cell lyate were the proteins from the cells. Then we mixed the LAS with the cell lyate. We then placed the tubes into this machine that determined the n/p ratio. Meanwhile, JM used excel to plot the data. The data ranged anywhere in between 5 million and 12 million cells for the different trials. We were looking for an increase of protein as the trials went on. And for once, we got a good result! The number of proteins did end up increasing. Then we repeated all of these steps five times to make sure that we obtained accurate data. Seung Ha and I both got turns doing this process. However, our data wasn't accurate because we produced a few bubbles when we initially pipetted which throw off the results. Then, JM taught us how to plot the data that we received. The process doesn't sound hard, but it was very tedious and took about the whole time. So this was it for today!
Sunday, November 6, 2011
Day 12
November 1, 2011
Seung Ha joined me today at the Med Campus. We started with Kellin explaining my project to her and showing her what I was doing. Not long after, JM arrived so we could do our interviews. Seung Ha and I interviewed our mentors simultaneously. It was very interesting and fun to learn more about each of them. The interview lasted longer than expected which was about 45 minutes. The most interesting fact that I found out about my mentor was that he used to comepetitvely dance in middle school and high school. The most interesting fact that I found out about Seung Ha's mentor was that he used to want to be a lumberjack all throughout his childhood. Once we finished the mentors' interviews, we interviewed Dr. Mao together. I asked the questions while Seung Ha wrote down his answers. He really focused on his family and how it was one of his main priorities. I found it funny and interesting how he claimed that he had "lost all of his creativity" ever since he became a scientist. We kind of had to speed up the last few questions since we were out of time.
Seung Ha joined me today at the Med Campus. We started with Kellin explaining my project to her and showing her what I was doing. Not long after, JM arrived so we could do our interviews. Seung Ha and I interviewed our mentors simultaneously. It was very interesting and fun to learn more about each of them. The interview lasted longer than expected which was about 45 minutes. The most interesting fact that I found out about my mentor was that he used to comepetitvely dance in middle school and high school. The most interesting fact that I found out about Seung Ha's mentor was that he used to want to be a lumberjack all throughout his childhood. Once we finished the mentors' interviews, we interviewed Dr. Mao together. I asked the questions while Seung Ha wrote down his answers. He really focused on his family and how it was one of his main priorities. I found it funny and interesting how he claimed that he had "lost all of his creativity" ever since he became a scientist. We kind of had to speed up the last few questions since we were out of time.
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