science – Page 3 – CA Work Experience Program

Day Six – EtBr Fun

Today I was back at the UNC Lineberger Cancer Center for some more adventures. I started off, like usual, talking to Dr. Bermek about the today’s experiments. There were many different new things she wanted to test because of some of the results from the experiments we had completed. The experiment we needed to get done first was using Ethidium Bromide (EtBr). We had tried to run the experiment using EtBr last week, but we discovered that EtBr only has a shelf life of about two years and the one Dr. Bermek was using had been in the lab for 13 years. When we analyzed the gel last week the results were not what they were supposed to be probably because of the expired EtBr, so today, we ran the same experiment with fresh EtBr. The reason we were using EtBr is to check for nicked DNA. Topoisomerase relaxes DNA (un-coils the supercoiled DNA strands) by creating an opening in the DNA, but based on a gel we had run last week with topoisomerase, we could not tell whether the nicked DNA got resealed or not which is what the EtBr would tell us.

Dr. Bermek had some samples being incubated so we spent the first hour talking and designing future experiments while we were waiting. There was lots and lots of math involved which threw me a bit of a curve ball, but I tried to keep up as best I could. The most interesting part was figuring out the calculations for reactions that needed the same final volume, but the samples were at different concentrations. There was a lot of dilutions involved. It fascinated me how fast and intuitive Dr. Bermek was when designing experiments. It was like she was creating a recipe. I am comfortable with carrying out a lot of the experiments we have done, but I will need much more experience in math and more practice to design my own experiments.

Once her samples had been incubated, we started on the EtBr experiment. Dr. Bermek prepared the samples because there were many ingredients while I prepared my first Agarose gel by myself. I measured out 0.5g of Agarose and added 150mL of TAE buffer. Then, I heated the mixture for 2 minutes and 30 seconds making sure to swirl the mixture every once in a while. Dr. Bermek taught me to run the Erlenmeyer flask (with my concoction) under cold water to cool the mixture faster. I also learned that we needed to add the EtBr to the gel and buffer instead of to the samples.

Once, I had finished making my first Agarose gel, I helped Dr. Bermek get the samples ready for loading by adding 4uL of 6x loading dye to each tube. Next, it was time for loading. I was a little more comfortable with loading Agarose gels, so I loaded the first lane and did pretty well; however, when I started loading the second lane, the tip of my pipette fell off and the sample went into the buffer. Luckily it was just the control, but after my mistake, Dr. Bermek loaded the rest of the samples because neither of us wanted to re-do the experiment for a third time. While we were completing the experiment, Dr. Bermek used a really cool heating mechanism that utilized little rocks instead of water, so people didn’t have to refill the container because rocks don’t evaporate like water. I thought it was a very clever idea.

Once the gel was running, Dr. Bermek checked some samples for their concentration of ATPase/phosphate. She used a really cool technique that I had never seen before. She used a plate with lots of different wells and added 180uL of water, 20uL of the samples, and 30uL of phosphate reagent. The samples and water were clear, but when she added the phosphate regent, the samples turned different colors. The shade of the bluish-green color of each sample indicated the amount of phosphate. I felt like I was experience a magic trick.

Towards the end of the afternoon after the samples had settled, we checked the concentration of the samples in a special machine. The machine gave us the exact concentration of each sample, but by that time, it was the end of the day. I’m excited to analyze the different concentrations as well as get the results of the gel we ran today when I come back tomorrow!

The magic color changing ingredient.

My first Agarose gel I prepared all by myself!

The clever heating mechanism.

The colored samples being loaded into the machine that checks their concentration of phosphate.

Day Five – Gaining Independence and New Accomplishments

After a nice restful weekend, I was ready for day five. Today I worked with Dr. Todd at CA. She had some of the proteins I was working on with Dr. Bermek. With Dr. Bermek I had been working with the protein UL8. Dr. Todd had different variants of the protein like UL30. We ran a protein gel with samples sent over from UNC to check them and see if they were clean samples. With Dr. Todd watching, I set up the Tetra gel box and gel apparatus ALL BY MYSELF (I was very proud to put my new skills to use). The only thing different was this time is instead of putting a plastic dam parallel to the gel, I put another gel opposite to the first gel because we needed two to fit all the samples. Once the gels were out of the refrigerator, I opened them, tore off the plastic on the bottom, removed the comb, put the gel very tight against the gasket, aligned the red and black stickers to the red and black on the gel box, poured the buffer in the between the gels, checked if it was leaking, poured the rest of the buffer into the box, and then just like that I had set up my first gel box all by myself. Once I finished my minor accomplishment, I prepared the samples. I added 4uL of 10x loading dye to each tube. This took quite a while because there were 16 samples. Then, I added 12 uL of each sample to the each tube that already had loading dye in it. After that, I put the samples in a hot water bath at 95 degrees Celsius for five minutes. Once the samples were nice and toasty, it was time to load them in the gels. Loading gels is very tricky, so I was quite nervous for this step. I didn’t want to mess anything up that would contaminate other wells because I didn’t want to re-prepare all 16 samples or cause any extra steps for Dr. Todd. Since Dr. Todd and Dr. Bermek’s gel setup was a little different, Dr. Todd showed me how to load by demonstrating on the first two lanes. I did much better loading the gel this time since I had a little bit more practice and the pipette tips were much narrower making the placement easier. I didn’t poke the gel, and each sample went into the correct lane. Just like preparing the samples, this process was very time consuming and intricate. Once I loaded all the lanes, I turned on the electric current and let the gel run for 50 minutes.

In the meantime, I helped Dr. Todd with some lab work that needed to be prepared for next year’s Biotech class. I helped organize, hole punch, and sort a list of all the primers that had already been designed with all the information about them. Then, I labeled tube after tube with the primer name and whether it was a forward or reverse primer. Each primer needed to have duplicates, so there were a lot of tubes. Once I had finished labeling the tubes, I pipetted 30 uL from the stock primers into the new labeled tube. This process was very tedious because there were a lot of caps to unscrew and a lot of pipette tip changes, but I was happy to be able to help Dr. Todd out. Once, I had finished, Dr. Todd gave me the good news that the protein gel worked out. We had no idea what the samples were like, so she was excited that they showed up on the gel. I was excited because I completed the whole process of running a gel by myself. I prepared the samples, prepared the gel box, loaded the samples, and ran the gel. It was a day full of firsts and new accomplishments.

Today was less hectic than the others days of the WEP because there were not four or five experiments going on like at UNC. It was nice to have a slower day, but I’m excited to return to the UNC Lineberger Cancer Center tomorrow for an action packed day full of new lessons and experiences.

All the samples I prepared to load into the protein gel.

The finished stained protein gel.

The Tetra gel box I set up all by myself with gels on both sides.

The gel loaded with samples.

All the tubes I labeled and added 30 uL of primers too.

Day Four – Exciting Results!

Today I felt much more comfortable and at ease. I no longer got lost when walking down the seemingly identical hallways to get to the Griffith Lab. I also ran into one of the other women who works in the same room as Dr. Bermek as I was walking in. She was really nice, and we had a short conversation as we were walking to the lab. It was a nice way to start the day. What I really like is all the other people working alongside Dr. Bermek are women. There is a great atmosphere in a place I thought would be strictly business. All the women I am surrounded by are extremely smart with many degrees. They all help each other out when they have questions about a protocol or need to borrow a pipette or gel lid. They also are always joking around with each other which I found really entertaining and funny. Once I walked in, Dr. Bermek and I, like every morning, made a plan for the day. I had a made a table of the amounts of supercoiled and relaxed DNA I counted on the EM on Tuesday. We took a look at the table and began calculating percentages of supercoiled vs relaxed DNA from the plain DNA sample, the UL8 sample, and the UL8 + CIP sample. I had a little bit of a hard time with the math at first but I caught on quickly. Once we finished calculating the percentages, we compared our results to the gel we had run the day before. The EM percentages were a good indicator of how the protein and CIP acted, but we also ran a gel because a lot of my counting and categorizing was personal based on which category I though the DNA would fall into. The results from the EM and the gel were pretty similar which was nice to see that I had counted somewhat correctly.

After we finished calculating the percentages of supercoiled vs. relaxed DNA, we checked on the agarose gel we had set up yesterday afternoon since it had finished being run and stained. The gel we ran was similar to another gel Dr. Bermek had already run, but she wanted more distinct bands, and on this gel, we tested time and concentration. Dr. Bermek thoroughly washed the gel with water because she knew she potentially wanted to use this gel for her paper, so she was very careful. Once Dr. Bermek saw the gel image she was jumping up and down with elation. She said it was one of the prettiest gels she had ever seen. The bands were really distinct which is exactly what she wanted. We were debating on whether to use a 0.6%, 0.8%, 0.9% or 1% gel, and we ended up settling on a 1% gel because of other protocols we had read online and it paid off. I, with a little bit of help from Dr. Bermek, made the gel that worked so well, so I was super excited. She told me “I need you here more often” which was a wonderful thing to hear. Dr. Bermek did not enjoy making agarose gels very much. When she showed the gel to one of the head guys in the lab, he also reiterated how nice the gel looked. Seeing the gel turn out so nicely made both Dr. Bermek’s and my day. We sat down and analyzed the gel. The basic conclusion we came too after a long conversation was the DNA began to relax with 75nM of the UL8 protein. With the reaction time gel we saw the protein began relaxing the DNA within only 15 seconds and the DNA became super relaxed in only two minutes.

After we finished analyzing the gel, we began to set up the next experiment. We were testing a drug used for the herpes simplex virus to see how it worked with the topoisomerase experiments we had been doing the past couple of days. Setting up the different amounts of each ingredient for the experiment required many calculations. I was nervous about all the math and didn’t fully catch onto everything. Each ingredient needed to be diluted which took at least 45 minutes to make all the dilutions. I helped Dr. Bermek with a couple of the dilutions, but she physically added all the ingredients into each tube. There were so many steps she let me sit down for a while and just watch her because of the complexity of the protocol. I am also much slower at pipetting than she is. Once she finished adding all the ingredients to the tubes, we had to wait awhile to incubate the samples. Dr. Bermek and I talked for a little while before it was time for me to go while we were waiting. It seemed like a little bit more of a relaxed day, but I still learned so much. It’s crazy how much knowledge I have obtained in just four days because I am immersed in each experiment. To me, working in a lab and learning about science is sort of like learning a foreign language. You have to immerse yourself and make mistakes in order to improve. I’m excited to rest up this weekend and recharge for another fun week!

The beautiful 1% gel I helped make being stained.

The wonderful results of the Agarose gel with very distinct bands.

Day 3 – Gels, Gels, and More Gels!

Day three was full of gels, gels, and more gels. I felt much more comfortable today due to my acquired knowledge about gels from biotech class. Even though I was familiar with the process for running gels, there were still some differences between our Biotech protocol and Dr. Bermek’s protocol which allowed for so much learning. Preparing the gels one on one also helped me learn and understand so many new things about them because I didn’t have any lab partners to rely on. I started the day similar to yesterday with a run down about the results from the experiments that were pending. Once I heard the results, we made a plan for today. The first thing we did was run a protein gel. I was vaguely familiar with this type of gel because of our recent fish project in biotech. I really enjoy Dr. Bermek’s warm and comforting personality. When I make a mistake she’s really nice about it. I really appreciate how she lets me feel like I’m involved with each experiment while giving me just the right amount of help I need.

First, I added 4 uL of 6x dye to tubes labeled 1-9. Then, I added 14 uL of each sample to the various tubes. Once the samples were ready to be loaded, she walked me through the process of setting up the protein gel. Unlike an agarose gel, the gel we used for protein electrophoresis was already prepared, so we didn’t have to pour it. We retrieved the ready-made gel from the refrigerator. We put on gloves for this specific gel because it needed to be handled with care. We took it out of its package and removed the sticky strip of plastic on the bottom or else the electric currents would not run through it. We also removed the comb from the top, so we could load the samples. Then, we placed the gel on one side of the tetra gel box. We made sure it was firmly placed into the gasket so there were no leaks. Parallel to the gel, we put a sheet of plastic so the liquid TBE buffer would be contained. When placing the piece of plastic I learned it was important to have the words “buffer dam” facing me. Dr. Bermek showed me some tricks when setting up the gel like putting it on the side closest to the edge of the gel box so the samples would be easier to load. Once we had the gel rig all set up, we placed it in the tetra gel box with the red cathode matching the red sticker on the box and the black anode matching the black sticker on the box. Once the whole box contraption was set up, it was time for loading the samples. I was extremely nervous because, for me, this was the hardest gel to load. The wells were so small and samples could easier drift into another well causing unwanted contamination. With Dr. Bermek’s wonderful teaching approach she showed me how to load the first three wells and then gave me a chance to do the last 7. I was doing okay on my first two, but when I got to the third one I didn’t place my pipette in at the right angle and some of my sample got into the next well. I informed Dr. Bermek of my mistake and she was really understanding a offered an easy fix. She pipetted out the contaminated well and finished loading the rest of the samples. Once they were all loaded, she turned on the electric currents. Unfortunately, 30 minutes later we saw the samples swimming in the buffer. We made a common mistake when turning on the electric currents because the black and red wires got mixed up. Since we realized 30 minutes into the gel being run, we couldn’t salvage the samples.

We repeated the experiment again, but this time, I did it all almost entirely by myself. I setup the gel rig and box under Dr. Bermek’s eye but I physically did everything. I then, repeated mixing 9 more samples, so they would be ready to load. I incubated them and spun them all by myself. Then while Dr. Bermek was checking on another gel. I loaded all 10 wells by independently with only a few small mistakes. I was very proud of myself. I won’t know until tomorrow how the gel turned out because it needed to be stained for a long time, but I’m hoping everything worked out well. Once we had finished repeating the protein gel, Dr. Bermek’s agarose gel she had done the night before had finished staining, and she showed me the results. The reason she did this gel was to compare the work I did with the electron microscope yesterday. She showed me the gel image and began explaining and annotating it. The first lane there was just plain DNA which was the control. It had a lower molecular weight, therefore it was super coiled. This gel confirmed our results we obtained from the electron microscope yesterday. The second lane contained the UL8 protein which relaxed the DNA therefore the band was at a higher molecular weight. The third band had UL8 and CIP. UL8 relaxes the DNA but CIP supercoils it, so the band had a lower molecular weight meaning the DNA was supercoiled showing us the CIP was working. The fourth lane had topoisomerase which again relaxes the DNA therefore the band was at a higher molecular weight; however, lane 5 did not produce results we wanted. Lane 5 had topoisomerase and CIP, but the band had a higher molecular weight meaning it was relaxed and not supercoiled even though CIP is supposed to super coil the DNA. We suspected that since the topoisomerase she used was a different brand than normal it might require more CIP than the UL8 needed. The next 6 lanes were treated with different amounts of UL8 which had all the bands at a lower and higher molecular weight meaning they were intermediate DNA (some parts supercoiled and some parts relaxed). It was cool to see what intermediate DNA looked like through the EM and on a gel. The last 5 lanes had topoisomerase and Ethidium Bromide. The topoisomerase relaxes the DNA whereas the Ethidium Bromide is supposed to super coil it, but again, our results didn’t quite match what we were expecting. I learned so much about the function different proteins have on DNA. It was cool to compare and contrast my findings on the EM and on Dr. Bermek’s gel. I like how in science you can do so many different tests and learn about the functions of things. After our day full of various gels, we topped it off setting up one more agarose gel to use for tomorrow. I really enjoyed today. I got to work with techniques I was familiar with, but still learn countless new things about gels. I also learned so much, just from, hearing about and annotating the gel image from yesterday. I don’t know what’s in store for tomorrow, but I can’t wait to get some of our results and continue preforming experiments.

Informative annotated Agarose gel.

Tetra gel box setup and ready to be run.

My beautifully loaded wells!

Our sad samples swimming in the buffer after our mistake.

Second times the charm! The correct box with the samples down at the bottom of the gel instead of floating in the buffer.

Day 2 – Exploring the Wonders of the Electron Microscope

Day two was just as action packed as the first. The anticipation from the first day was slowing wearing off, and I felt ready to work. I started off the morning hearing about the results from the previous days experiments which led to the plan for today’s work. Almost immediately after my arrival, I headed into the EM (electron microscope) room with Dr. Bermek. I had gotten a taste of the EM yesterday, but today it was like I was eating a three course meal. Dr. Bermek loaded a grid which required lots of precise and intricate detail to move the spec sized grid, put it in the right place on the tip of a long metal wand looking piece of equipment, and put the wand shaped piece of metal into its correct spot on the EM. Since it took a little while to get loaded she showed me how to switch between the two computers in front of us which made my task a little more daunting because I had to worry about working with two different programs.

Once the EM, was loaded she showed me how to focus the beam – another tricky aspect of the EM. There were so many controls it felt like I was sending a space ship into outer space. She first showed me the magnification which was pretty self-explanatory except it worked in accordance with another control – the intensity. Whenever I changed the magnification I also had to change the intensity. I had to make sure not to move the magnification to quickly or too much or the beam would be too wide for the etched circle on the plate, and I wouldn’t be able to see anything. The next control she taught me was the one that allowed me to look at different parts of the grid. There was also a button called R1 which I would press when I was finished choosing the part of the grid I was looking at, and it moved the bottom of the plate upwards so I see the sample we were analyzing. The first sample we were looking at was the DNA which was the control. My job was to analyze the sample by counting the number of super coiled, relaxed, and “other” stands of DNA that appeared. After showing me the basics of the EM controls and the features on the computer, Dr. Bermek left me to count while she continued working on a different experiment. I was a bit hesitant when she left because I didn’t trust myself with a machine that costs more than college tuition, but she had set up everything so well I felt prepared, and I was very cautious. As one sample after another went by I felt more comfortable. I used a handy counting machine that made the process a little easier because on some samples I was categorizing over 400 DNA strands. The hardest part was figuring out where I was on the sample because it all looked the same, and I didn’t want to accidently count a DNA strand twice. It was really neat to see up close what super coiled and relaxed DNA look like. Some grids had a UL8 protein while others had topoisomerase. The different solutions added caused the super coiled DNA (what its normally like) to become more open looking by uncoiling it.

Once I had finished counting the results from mini grids, Dr. Bermek asked me to drop spread some more grids. It made me feel really important because she left me to do it by myself while she worked on something else. I used the same technique from yesterday where I dipped each grids in various solutions and then immersed it in 75% ethanol for 30 seconds and then 80% ethanol for 45 seconds. Once I was done preparing the samples, we looked at a few more things through the EM, and just like that my second day was already over. It was super cool to work so closely and independently with a electron microscope, a piece of material super intricate but really useful. I was also glad I could help Dr. Bermek with the preparation for other experiments. It felt like I was really working in the lab. Day two was just as informative and action packed as the first. I can’t wait for tomorrow!

My handy dandy counting tool.

Examples of what some of the DNA strands I counted looked like.

My Drop Spreading layout.

Up-close example of the tiny grids after they were prepared.

My control panel for working and manipulating the EM.

Day 1 – First Day in The Lab!

I started off the day greeted by Dr. Bermek in front of the UNC Lineberger Cancer Center. She escorted me into the building and introduced me to others working in the lab. I got to work quickly and acclimated my Biotech skills with a DNA mini prep for three enzymes PUC19, PGLO, and P-Amalyse for the next years Biotech class. The protocol I used was striking similar to those I had seen in ADV Biotech. I suspended the DNA pellet in Buffer P1 using my well-practiced pipetting techniques and continued the protocol that called for a centrifuge. This piece of equipment I had used many times before, but it was interesting to learn something new about it. I learned that when setting the centrifuge to the correct speed I needed to use the g speed (e.x 17,500 x g) instead of the rpm (e.x 12,000 rpm) because each centrifuge has a different rpm but the g speed is the same. After completing the protocol Dr. Bermek and I used a Qubit Fluorimeter to check the concentration of the samples. The Qubit was a new piece of equipment I had never seen before, but it was simple, easy to use, and much smaller than the spec machine I had worked with in Biotech. Two of our samples, PGLO and PUC19 turned out well with a concentration around 50 ng/uL which was really awesome because Dr. Todd could keep them for next year’s class. Dr. Bermek and I could also use the PGLO for a future experiment. It was really nice that the first experiment I did at the Griffith Lab worked.

Next, I got to run some samples on a Agarose gel. I pipetted 6uL reactions on parafilm. Each reaction consisted of 1uL of the samples, 1uL of 6x dye, and 4uL of water. Once the 12 samples were pipetted onto the parafilm, Dr. Bermek and I loaded the samples into the gel box. I had run many gels in class before but I got to learn how to use and load a new system. Our gel also turned out successfully which was wonderful to hear, and the results we obtained are going to be helpful for future experiments. One of the coolest parts of my first day was getting to learn about the Electron Microscope or EM. The machine was very intimidating and marvelous at the same time. One of the people working in the lab was a major expert on the EM, and he was in the middle of fixing it when I arrived. To even be able to put samples in the EM there was a complicated process to prepare the samples that I got to help Dr. Bermek with. We used tiny grids (much smaller than a penny) that required special tweezer looking things to be able to handle them. We put solutions on parafilm and then dipped the grids into each one. Next, we placed the grids in one solution for 30 seconds and then quickly placed them in a different solution for 15 seconds one by one. To further prepare the grids, we used a complicated special machine that coated the grids in carbon and tungsten. I was easily impressed by the steaming liquid nitrogen used to cool down the machine. This process was intricate because the right amount of both carbon and tungsten had to be exact or we wouldn’t get proper results. I ended the day by looking at the images we obtained on the EM. I got to see single stranded, super coiled, and relaxed DNA up close which was magnificent. Overall, it was an action packed first day with lots of success. Working in a new lab was a little bit like the first day of Biotech class. There was a lot of new vocabulary, sciencey jargon, and math equations that were a bit challenging, but I got to learn so much on just my first day!

Working on my first experiment with Dr. Bermek.

Image result for electron microscope

The daunting Electron Microscope.