December 7, 2011
Today we did an experiment but I'm not exactly sure what the name of it was. We've done it before. It was a little tedious but it was interesting. The goal was the take this positively charged nanoparticle and place it in a cell. The significance of this process was that the nanoparticle was a polymer with DNA as opposed to a neutral polymer. This nanoparticle is able to produce a lot more Luciferase. Luciferase is apparently good for the body which was why it was the goal for the experiment. Then we added on to that experiment to calculate the data of how much luciferase was actually produced. We started with 100ml of Luciferase. Then we added 20ml of DNA, mixed the two together, and then placed the tube that contained the mixture into this machine that quickly calculated the amount. We did several trials. But all the trials ended up for some reason not working. Every trial showed a very miniscule amount of Luciferase which was not supposed to happen. JM thinks that maybe the cells had gone bad somehow. He said that the cells originally looked strange in the beginning, so that might have been the reason why the experiment didn't work. Of course it's a little upsetting when the experiment that you're working on for hours doesn't work, but it's unavoidable and it will continue to happen. That was pretty much all we did today.
Wednesday, December 7, 2011
Tuesday, December 6, 2011
Day 20
December 6, 2011
It was a very interesting day! We started with cell culture. I've been this before also in my Wednesday lab. It was interesting that they use similar techniques for completely different purposes. It was fun because Kellin let me do the entire procedure while he just told me what to do. We put 2mL of media (a mixture of chemicals and cells) into a tube and then placed it in an incubator for 10 minutes. Then we placed the tube in a centrifuge. The centrifuge rapidly spins the tube around which somehow makes all the cells settle to the bottom of the tube. We brought the tube back into the hood and removed the remaining solution but left the cells in the bottom. We placed 4ml of new media into the tube with cells and pipetted through several times to chop the cells and mix them with the media. Then we moved the solution into a different container that we could place under the microscope. Once we finished that, we made another gel gradient. While we were waiting for the gel to harden, he and the rest of the lab were just playing around with dry ice. He showed me what would happen if he put a frozen cube of CO2 in room temperature water. It boiled and looked like fog. It was really cool. Then he explained that if you place a small amount of water in a syringe and reduce the pressure enoughm the water will boil at room temperature! He attempted trying this but ultimately failed. He told me that boiling points differ depending on the altitude. In Denver which is high in the mountains, water has a lower boiling point and it takes a longer amount of time to boil something there. Then, Ms. Rein came to visit my lab for a few minutes. But it was ashame because we had already finished everything we wanted to do that day, so Ms. Rein didn't get to see me do anything.
It was a very interesting day! We started with cell culture. I've been this before also in my Wednesday lab. It was interesting that they use similar techniques for completely different purposes. It was fun because Kellin let me do the entire procedure while he just told me what to do. We put 2mL of media (a mixture of chemicals and cells) into a tube and then placed it in an incubator for 10 minutes. Then we placed the tube in a centrifuge. The centrifuge rapidly spins the tube around which somehow makes all the cells settle to the bottom of the tube. We brought the tube back into the hood and removed the remaining solution but left the cells in the bottom. We placed 4ml of new media into the tube with cells and pipetted through several times to chop the cells and mix them with the media. Then we moved the solution into a different container that we could place under the microscope. Once we finished that, we made another gel gradient. While we were waiting for the gel to harden, he and the rest of the lab were just playing around with dry ice. He showed me what would happen if he put a frozen cube of CO2 in room temperature water. It boiled and looked like fog. It was really cool. Then he explained that if you place a small amount of water in a syringe and reduce the pressure enoughm the water will boil at room temperature! He attempted trying this but ultimately failed. He told me that boiling points differ depending on the altitude. In Denver which is high in the mountains, water has a lower boiling point and it takes a longer amount of time to boil something there. Then, Ms. Rein came to visit my lab for a few minutes. But it was ashame because we had already finished everything we wanted to do that day, so Ms. Rein didn't get to see me do anything.
Sunday, December 4, 2011
Day 19
November 30, 2011
Today was more of a boring day. I didn't get to do any hands-on things. Seungha and I only watched him do a few experiments. He didn't really have much planned out for us, so he tried to think of a lab for us to do. So he tried to set up, but the equiment was occupied by others for most of the time. So he decided that he would show us autoclaving again, which is a machine that sterylizes pipet tips. But when we arrived at the autoclaving room, the machine was already in use. So we went back to the lab and discussed the final presentation for a little while. He tried to plan out when we should start working on the presentation and how he will help us. The problem was that I'm not even doing the presentation on my Wednesday project so he's not going to be very helpful to me. But this got me thinking about when I should start working on my presentation and talking to Kellin about what I should do. This was really all we did that day so he let us out early. We walked to a cafe in Latrobe Hall and just stayed there until it was time to go.
Today was more of a boring day. I didn't get to do any hands-on things. Seungha and I only watched him do a few experiments. He didn't really have much planned out for us, so he tried to think of a lab for us to do. So he tried to set up, but the equiment was occupied by others for most of the time. So he decided that he would show us autoclaving again, which is a machine that sterylizes pipet tips. But when we arrived at the autoclaving room, the machine was already in use. So we went back to the lab and discussed the final presentation for a little while. He tried to plan out when we should start working on the presentation and how he will help us. The problem was that I'm not even doing the presentation on my Wednesday project so he's not going to be very helpful to me. But this got me thinking about when I should start working on my presentation and talking to Kellin about what I should do. This was really all we did that day so he let us out early. We walked to a cafe in Latrobe Hall and just stayed there until it was time to go.
Tuesday, November 29, 2011
Day 18
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!
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!
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.
Sunday, October 30, 2011
Day 11
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.
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.
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.
Tuesday, September 27, 2011
Day 1: Solar Decathlon
First time writing in the blog! So on Monday we went on a field trip to the Solar Decathlon. I did not know what to expect at all, and I'm not interested in the whole "going green" concept but I surprisingly found it very interesting and entertaining. Each house was built in a different way that not only made it helpful to the environment, but also conserved space. Something that grabbed my attention in my of the houses was the bedroom. Some houses had the typical bedroom. But many others found a way to put a decent sized bed into a small knook in the house such as above the TV, inside the wall, or even inside a desk! However, even though many of these ideas help the environment and conserve space, it requires much more human energy. For example, if the bed was stored in the wall, it would take much work to set it up every night and fold it back up every morning. This can be a tedious project to do everyday, so this might turn people against the whole idea.
Even though I was amazed at all of the houses, my favorite was The First Light which was the house created by the Victoria University of Wellington in New Zealand. Not only was it built to conserve energy, but it was also very livable. The numerous large windows made the house seem very open and full of light. Surprisingly, the bathroom seemed very nice as well. The flooring of the bathroom was just bamboo, and underneath were plants that the excess water drained into. Even though I found this particular house very interesting, I really enjoyed walking through all of the houses and learning about the different ways to help the envirnoment.
Even though I was amazed at all of the houses, my favorite was The First Light which was the house created by the Victoria University of Wellington in New Zealand. Not only was it built to conserve energy, but it was also very livable. The numerous large windows made the house seem very open and full of light. Surprisingly, the bathroom seemed very nice as well. The flooring of the bathroom was just bamboo, and underneath were plants that the excess water drained into. Even though I found this particular house very interesting, I really enjoyed walking through all of the houses and learning about the different ways to help the envirnoment.
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