simonsaysrht12-blog

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rs35716097 (the Blue SNP)

Entry #12

This was the problematic SNP that I could not design a primer for. There was something that me and Libby, one of my wonderful lab mentors, could not figure out. However, after Steve got his hands on it, he had no problem designing the primers that were to cut off that 20 base pair region that is required like the previous SNPs. This SNP, based off of Brooke’s data, is actually pretty interesting. In her list of candidate SNPs, Brooke found that it is part of the gene that is associated with osteoporosis. By learning this I realized the complexity of genetic makeup, and how progressive science is. The solution of curing one auto-immune disease may lead to the cure of another auto-immune disease if we know where to look. 

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rs2705618 (the Green SNP)

Entry #11

Above is another one of the 3 SNPs that I have chosen to undergo methylation analysis on the Q24 machine. This SNP was chosen from a list of candidate SNPs that a previous fellow wanted to analyze. The information left by the previous fellow indicated that it was located in chromosome 4. Anyway, after choosing this SNP I had to locate where and what kind of base pair existed at its location. Upon looking at dbSNP, a database that had this kind of information, it was at a CpG site that had a Cytosine paired with a Guanine. Then, I had to select a region around the SNP that was about 20 base pairs apart in both directions. This region is going to be cut out by primers and it is also what is going to be amplified in the pyrosequencing process. Later in the process I named this the Green SNP because I like color-coding and I figured that I am going to have to identify which samples were being tested for the methylation of this specific SNP, so the color green was easy to remember than rs 2705618.

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rs13136820 (the Purple SNP)

Entry #10

Above is one of the 3 SNPs that I have chosen to undergo methylation analysis on the Q24 machine. This SNP was chosen from a list of candidate SNPs that a previous fellow wanted to analyze. However, there was very little information about this SNP left by the previous mentor. All she said about this SNP was where it was located on the human genome (LOC101060498) and the fact that it was on her list means that it was around a location that was known to play a factor in the development of multiple sclerosis. Anyway, after choosing this SNP I had to locate where and what kind of base pair existed at its location. Upon looking at dbSNP, a database that had this kind of information, it was at a CpG site that had a Cytosine paired with a Guanine. Then, I had to select a region around the SNP that was about 20 base pairs apart in both directions. This region is going to be cut out by primers and it is also what is going to be amplified in the pyrosequencing process.

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January 27 Entry #9

“I’m so done” - Me

Choosing three SNPs was harder than I thought. The first time I had to choose my SNPs from a documented list of “Candidate SNPs” that a former fellow named Brooke ran some data on. I had to make sure that they were somehow related to the development of MS, check its Minor Allele Frequency (which I don’t understand completely myself), the surrounding sequences around the SNP, where it is located, and the kind of transition it was. I had to use sites such as Ensembl, PubMed, and Taqman to retrieve all of this data. Now the frustrating part came from having to check if Brooke has already genotyped these SNPs, and trying to come up with information about the SNP. But here comes the discouraging part: when I finally made my list of three SNPs, I found out that I had to find them in another table that shows them on a list of samples, but the worst part is that two of my SNPs did not show up on the table. What I had to do then was to choose some other SNP from this new table and just go on from there. If I was going to do that, why didn’t I choose from this new table in the first place!? Eventually I found another one but I ran into another problem. I had to make sure that all these SNPs were making CpG sites through the Q24 program I had to install, and one of them did not show any creation of CpG sites, and that was one of my original SNPs. So, by the end of the day, the three original SNPs that I worked so hard for ended up being useless and I basically chose my current SNPs from this new table with little to no research on them.

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January 13 Entry #8

Did I mention SNPs?

SNPs is the acronym for Single Nucleotide Polymorphisms. SNPs are variation in a single base pair in a DNA sequence. These variations are very common and in fact, about 10 million SNPs happen in the human genome (U.S. National Library of Medicine). An example of SNP can be when a nucleotide of cytosine is replaced by a nucleotide thymine in a certain strand of DNA. These variations help researchers determine a lot of things such as an individual’s susceptibility to environmental factors and their risk of developing a certain disease. In context of my independent study, SNPs play a key role in studying the development of Multiple Sclerosis. It is suspected that DNA methylation is related to the epigenetics of MS, and the methylation of DNA usually happens at CpG sites. CpG sites are places along a strand of DNA when a Cytosine is followed by a Guanine, and it is suspected that the higher the concentration of CpGs, the higher the chances that DNA becomes methylated. Because SNPs are occur at a single nucleotide level, they can create CpG sites which affects the methylation of DNA, and thus affects the development of MS.

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January 9 Entry #7

SNPs SNPs and more SNPs

After winter break, things in the lab are beginning to look more like lab work. We are all undergoing the process of starting our individual projects and I am going to be working with SNPs (Single Nucleotide Polymorphisms) of Multiple Sclerosis patients. Under the guidance of my mentor Libby and some data from a previous fellow named Brooke, I am supposed to choose some SNPs, create primers for it, amplify them using fancy machines that I will go into depth in a later journal entry, and then make conclusions about these SNPs that I chose. However, I have found it difficult choosing these SNPs because I have to make a list on 10 SNPs in prioritized order. In other words, I have to explain why I chose these SNPs. It may seem easy at first but considering that I have to look at data bases with intense vocabulary, such as PubMed,  I am having a hard time deciphering the significance of these SNPs. Luckily, I just have to complete 3 for now, but this is going to take some time and prayer. 

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December 9 Entry #6

“Hey. How’s life?” -Steve

A lot of issues has happened in between the making of this entry and last one. I managed to finish writing that paper on time, and I sent it to Ms. Sauer, however it turns out she never received it via email, so I had a big fat 0 on my progress report sitting there without me having any idea. I don’t see Ms. Sauer that often, and I suppose it is still my fault probably because I did not type the correct email address due to the amount of stress I was under. I can breathe a little better now since I handed her a physical copy so that everything is in her hands. Now with that paper out of the way my life has been slowly settling down back to normal, however there is still plenty to catch up with. I hope these blog posts will begin to become more filled with scientific data, pictures of lab results, and less rants. And I hope I can catch up with Maya and Sam, because as I am writing this, Steve and Maya are going over computer programs that we will be using in the future to record and analyze data. It’s time to get serious....so help me God.

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November 18 Entry #5

I COULDN’T DO THIS

This review is atrocious. I am actually crying for multiple reasons. My first reason is that even though I dedicated an entire day to write this review I still could not do nothing more than just readjust my outline. I spent the whole day at the library from 9am to 9pm and still I could not write anything that I was satisfied with. It has been a number of days since it was due and still nothing is getting on paper which brings me to a second reason to why I am crying: Ms. Sauer gave me mercy. I am so thankful that she has given me an extension up to this day, so it is my duty to finish it. The final reason for my current state of depression is the disappointment I have for myself due to the fact that I am unable to come up with a clear thought process. All I have is a decent outline and some pretty dense articles, but creating something out of it is taking way too long to do. I just want this over with, so I’m just going to turn in whatever I have.

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I CANT WRITE THIS PAPERRRRR aweawidvbgqerjfbqadrjifbqlrgIWEJGQLK 

#i'm dead

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November 4 Entry #4

My Birthday at the Lab

It’s the 4th of November which also happens to be my birthday...which was semi-exciting I guess. Libby, one of my lab mentors, baked me some brownie-marshmallow-chocolate-pastry that I have no idea what to call it, but it was a Pinterest recipe that I am very thankful for. Nothing really lab intensive has happened since the last entry, however there is an encroaching literature review that is due on the 14th of November. I have absolutely no idea how to address this review because I have not done anything of the sort. I have gotten a lot of sources from PubMed and other articles that Steve has given me, but putting it all together in the format of the rubric is foreign. I am concerned that I will not be able to write anything decent simply because I know that writing has never been my strong suit. Maya and Sam seem to have a gist of what to do but I just feel helpless. I’ve spoken to Steve a number of times, and every time I am face to face with him the project seems clear, but when I go home and sit in front of my computer I cannot muster up a clear thought process. I understand that this literature review is supposed to reflect my understanding of the project that I have been assigned, and now I feel like I don’t understand anything at all, or at least the big picture. I know what multiple sclerosis is. I know that we are studying it because there is no known cure of the disease. I know that epigenetics somehow play a role in the development of multiple sclerosis. I know that my job at the lab is to specifically focus on how MS is affected by DNA methylation. From the literature that I have analyzed and gathered it seem that the genetic study of MS and what the DMPI is doing is nothing new, but we are painting a portion of a puzzle piece that belongs to a bigger picture which belongs to an even bigger picture. I hope that I can write something that makes sense, so help me God.

#help #i need somebody #literature review

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October 21 Entry #3

How To Make Agarose Electrophoresis Gels Using Gel Red

Consumables:

- 1% Agarose Gel

- LE Agarose (.50g)

- TAE Buffer (50.00ml)

- Gel Red (2.00uL)

Process:

1) In an Erlenmyer Flask, swirl and mix TAE Buffer and LE Agarose

2) Microwave flask for 30 second increments, swirling flask between each.

*The solution is ready when agarose crystal beads are no longer visible*

3) Cool solution in a pot of water while swirling for 1-2 minutes

4) Add Gel Red Stain and swirl gently for at least 30 seconds

5) Pour contents into a mold and remove bubbles with a pipet tip.

6) Add combs into the mold and allow it to set for 20 minutes

7) Remove the combs and add 2uL ladder into the ladder lane

8)Pipet the required uL of 2x loading dye on a piece of parafilm

9) Pipet DNA on top of the loading dye and mix well

10) Pipet DNA loading-dye mixture into the gel wells

11) Run the gel for 25 minutes at 90V

12) Visualize the gel on the gel imaging station

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Gel Imaging Station

#made with tumblr #gif

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October 7 Entry #2

“and the little babies are like falling into the ground” –Steve

In the next step of this scientific journey with my independent study with the DMPI, we performed gel electrophoresis. Gel electrophoresis is a method used to separate the mixtures of DNA, RNA, or proteins based on their size. Specifically, we used this method to determine whether or not the process of amplifying a sequence in our bisulfite-converted DNA sample was done correctly. The gel electrophoresis would have to show us strong, colored bands to indicate that we have a good sample to work with before we put it into the Pyro Mark Q24 machine. Unfortunately, we did not see the strong bands that we desired, but the issue was because the sample of unconverted DNA was already questionable from the beginning. My savvy mentor, Stephen Siecinski, suspected this issue knowing that the sample was old, so now he needs to get a hold of a better sample for us next time. Despite our unfortunate results, it was interesting to perform gel electrophoresis for the first time. I learned the basic process of making the gel itself, using the lab equipment that utilizes the gel, and understanding what the image of the gel with the colored bands tell about the sample. The quote above was when Steve explained how size determines how fast a band moves through the gel. The bigger the sample the slower it travels; the smaller the sample, the more quickly it moves through the gel. In addition to this post, I will follow up with an in-depth process of how we made and used the gels and images we took of the colored bands.

f4���

#science #gel electrophoresis #dna

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September 23 Entry #1

“I don’t like my children starving” –Libby

I arrive at the Duke Molecular Physiology Institute laboratories after my chorus class which is at the time when my lunch period begins. As excited as I am to begin this scientific journey with the DMPI, I am rather concerned with the fact that it interferes with my usual eating time.

 I try not to think about it too much, but it was quite distracting today when I was learning how to carefully pipet 737.15 microliters of water into a sample tray while my stomach would growl every 10 minutes. What is even worse is that when my blood sugar is too low my hands will begin to have tremors, so pipeting on an empty stomach has been my first obstacle that I have encountered so far during this experience. But luckily, one of my wonderful mentors named Libby and her motherly nature that I am so grateful for, had given me some delicious sustenance to ease my internal sufferings.  Above all, I am glad to say that all online training courses have been completed, and that I am so happy because now I am able to touch things in the lab and take my embarking steps into this scientific journey. 

#science #hungry #research #pipet #laboratory