Independent Study: Cognitive Disorders Lab Research
For today, I was focused on doing the second part of the experiment. I wasn't able to get any pictures because it was an extensive process. I had to pay close attention to detail and I didn't want to worry about my phone and any possible contamination that I would have to deal with when doing the PCR. I thought the process was fairly complicated and had to go slower than what it would normally be. Below is the handout that I used for both the RNA extraction, Reverse Transcription (Day 3), and then the PCR (note that I only put in the sections that I used).
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Today, I did RNA Extraction and Reverse Transcription. I decided that instead of writing out all of my steps, I would insert a time lapse video (considering that it took over two hours) to show what we did and the handout that we followed during the process. This took roughly the whole morning and when I had finished and finally got my cDNA, they moved on to show me what aliquoting was. While it wasn't directly tied to what I was doing in the morning, there wasn't enough time for me to start the second part of doing the PCR on the cDNA. The main focus of today was to watch and prepare myself for the experiment that I will be conducting on Friday. Polymerase chain reaction, also known as PCR, is a technique that is used to amplify small segments of DNA. For Friday, we will be doing Real-Time PCR on RNA. However, today I was soley focused on learning how to extract and isolate the RNA then reverse transcribing it into cDNA (which can be considered the prep that eventually leads to the PCR). Tomorrow the goal is to do the prep for the PCR, the extraction and reverse transcribition, so today I needed to familiarize myself with the process.
The first part of the day, I was reading over the protcol. I was given multiple links to several websites and different articles to read that was simply focused on PCR. While some of the articles breifly mentioned the extraction and reverse transcribition of the experiment, it wasn't fully explained until the afternoon where I saw the whole process of going to RNA to cDNA. Without stopping, it usually takes about an hour and a half to complete, but because the Lab Manager/Research Associate was explaining the instructions and with all of our questions, it took nearly two hours. Personally I thought that the amount of information was quite overwhelming. Even with my resources and the Lab manager explaining the the entire process, there was too much information that I felt I needed to retain. I expressed my thoughts about this and was reassured that I would still have my papers and that she will be guiding me along the way. Intitially, the fact that I was still confused caused some stress, but I realized that if I aksed questions throughout the process tomorrow I will have a better understanding of what I'm doing and the reasoning behind it. Today's goal was to become more comfortable in the lab setting and get accustumed to what I will be dealing with for the next three weeks. For my future projects, it was explained that I needed to have very precise amounts of a certain substance in order to have the least amount of variants and the more practice I had, the better off I would be. Not to mention the idea that they are building my confidence with certain tasks so that I am able to reach a point where I can become more independent towards my study, without needing the constant supervision. For this reason, they put me through a 'pipette bootcamp' where I would set the pipettes ot their highest setting and pipette water into a weight tray on a scale. I would record the weight in mg (1mg=1ul) and repeat 10 times per pippete. There were 4 different types of pipettes, each having the capacity to measure different amounts of microliters. After all of the data was gathered, I record the mean, standard deviation, and coefficent of variant. The link below will direct you to all of my raw data: https://docs.google.com/spreadsheets/d/1nASqhfpMdqDbUgmxk3EpmxX4fGoAritEaZNIY7SI1ag/edit?usp=sharing The process took quite some time due to the fact that I needed to work slowly to ensure that I was being as percise as possible. Looking back to my data and after calculating the coefficient of variant then turning it into a percentage, I found that a lot of my percentages were below one (you want the lowest percentage possible; the higher percentage, the higher percentage of varibility). This impressed me considering it was my first time. However, when I was measuring the p20s I had noticed that my numbers were a little off. After discussing my findings, I learned that the scale was inaccurate when weighing such a small amount of water. Despite this, I had a great experience and was excited when I saw that the numbers were getting closer to what they supposed to be.
These are the two graphs based on my data (the only difference is the scale). |
Welcome to my first independent study!
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